Trans-4-hydroxycyclohexyl phenyl amide mitofusin activators and methods of use thereof

ABSTRACT

Compounds and compositions including stereoisomers of 6-phenylhexanamide derivative small molecule mitofusin activators are described. In particular, mitofusin activators comprising derivatives of (trans-4-hydroxycyclohexyl)-6-phenylhexanamide, which are useful for treating diseases or disorders associated with a mitochondria-associated disease, disorder, or condition such as diseases or disorders associated with mitofusin-1 (MFN1) and/or mitofusin-2 (MFN2), or mitochondrial dysfunction, are described. Methods of treatment and pharmaceutical formulations are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/935,557, filed on Jul. 22, 2020, which is acontinuation-in-part of International Patent ApplicationPCT/US2020/014784, filed on Jan. 23, 2020, which claims the benefit ofpriority under 35 U.S.C. § 119 from U.S. Provisional Patent Applications62/797,513, filed on Jan. 28, 2019, and 62/949,060, filed on Dec. 17,2019, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants R41NS113642and R41NS115184 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofnovel active trans-4-hydroxycyclohexyl phenyl amide stereoisomers ofsmall molecule mitofusin activators and methods of use thereof.

The present disclosure generally relates to compositions and methods fortreating genetic or acquired central and peripheral neuropathies,neurodegenerative diseases, disorders, or conditions.

SUMMARY

Among the various aspects of the present disclosure is the provision ofnovel functionally active stereoisomers of small molecule mitofusinactivators and methods of use thereof.

One aspect of the present disclosure provides for methods of treatingneuropathies, neurodegenerative diseases, disorders, or conditions. Insome features, the method comprises administering to a subject atherapeutically effective amount of a composition comprising one or moreactive stereoisomers of mitofusin activators; the mitofusin activatorsstimulate mitochondrial fusion and subcellular mitochondrial transportin neurons, thereby evoking resistance to neuronal injury, acceleratingneuronal repair, and promoting neuronal regrowth and regeneration.

Another aspect of the present disclosure provides for a method ofactivating mitofusin in a subject in need thereof. In some features, themethod comprises administering to a human subject or affected animal acomposition comprising one or more active trans-4-hydroxycyclohexylstereoisomers of mitofusin activators; the activetrans-4-hydroxycyclohexyl stereoisomers of mitofusin activatorsstimulate mitochondrial fusion and subcellular mitochondrial transportin neurons, thereby evoking resistance to neuronal injury, acceleratingneuronal repair, and promoting neuronal regrowth and regeneration; thesubject or affected animal has a genetic or acquired central orperipheral neuropathy, neurodegenerative disease, disorder, orcondition.

Another aspect of the present disclosure provides for methods ofpreventing, mitigating, reducing, or enhancing recovery from iatrogenic,traumatic, or collateral nerve damage in a subject in need thereof. Insome features, the method comprises administering to a subject acomposition comprising one or more active trans-4-hydroxycyclohexylstereoisomers of mitofusin activators; the active stereoisomers ofmitofusin activators stimulate mitochondrial fusion and subcellularmitochondrial transport in neurons, thereby evoking resistance toneuronal injury, accelerating neuronal repair, and promoting neuronalregrowth and regeneration; the subject or affected animal has a geneticor acquired central or peripheral neuropathy, neurodegenerative disease,disorder, or condition.

In some aspects, the active trans-4-hydroxycyclohexyl stereoisomers ofmitofusin activators have substantially better functional potency thanboth 1-[2-(benzylsulfanyl)ethyl]-3-(2-methylcyclohexyl)urea (Cpd A,Rocha Science 2018) and2-{2-[(5-cyclopropyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta[b]thiophene-3-carboxamide(Cpd B, Rocha Science 2018).

In some aspects, the active trans-4-hydroxycyclohexyl stereoisomers ofmitofusin activators have substantially better drug-like pharmacokineticproperties than both1-[2-(benzylsulfanyl)ethyl]-3-(2-methylcyclohexyl)urea (Cpd A, RochaScience 2018) and2-{2-[(5-cyclopropyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta[b]thiophene-3-carboxamide(Cpd B, Rocha Science 2018).

In some aspects, the active trans-4-hydroxycyclohexyl stereoisomers ofmitofusin activators: target mitofusin-1 (MFN1) or mitofusin-2 (MFN2);increase mitochondrial elongation by enhancing mitochondrial fusion;enhance mitochondrial function measured as inner membraneelectrochemical polarization; enhance mitochondrial transport in nerveaxons; correct cell and organ dysfunction caused by primary or secondarymitochondrial abnormalities; reverse mitochondrial defects (e.g.,dysmorphometry, clustering, loss of polarization, loss of motility);restore, activate, regulate, modulate, promote, or enhance the fusion,function, tethering, transport, trafficking (e.g., axonal mitochondrialtrafficking), mobility, or movement of mitochondria (in, optionally, anerve or a neuron); enhance mitochondrial elongation or mitochondrialaspect ratio; disrupt intramolecular restraints in MFN2; allostericallyactivate MFN2; and repair morphological and functional defects indiseased or damaged neurons with mitochondrialabnormalities.

In some aspects, the active trans-4-hydroxycyclohexyl stereoisomermitofusin activator comprises one or more compounds having structuresrepresented by formula (I):

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereofwherein, R¹ may be a non-, mono-, or poly-substituted C₃₋₈ cycloalkyl,C₃₋₈ heteroaryl, C₃₋₈ aryl, or C₃₋₈ heterocyclyl.

In some aspects, the active trans-4-hydroxycyclohexyl stereoisomermitofusin activator may be a compound having a structure represented byformula (I)

wherein R¹ may be one of the following moieties:

In some aspects, R¹ may be optionally substituted by one or more of:acetamide, C₁₋₈alkoxy, amino, azo, Br, C₁₋₃ alkyl, carbonyl, carboxyl,Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl,F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/orthiophene; and optionally further substituted with one or moreacetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl,cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F,halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone,and/orthiophene. Optionally the aforementioned alkyl, cycloalkyl,heteroaryl, heterocyclyl, indole, or phenyl may be further substitutedwith one or more of the following: acetamide, alkoxy, amino, azo, Br,C₁₋₃ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O,phenyl, S, sulfoxide, sulfone, and/or thiophene.

In some aspects, formula (I) may be one of the following moieties:

Yet another aspect of the present disclosure provides for apharmaceutical composition comprising an activetrans-4-hydroxycyclohexyl stereoisomer mitofusin activator, optionallyin combination with one or more therapeutically acceptable diluents orcarriers.

In some aspects, the pharmaceutical composition comprises apharmaceutically acceptable excipient.

In some aspects, the pharmaceutical composition comprises one or more ofthe following: neuroprotectants, anti-Parkinsonian drugs, amyloidprotein deposition inhibitors, beta amyloid synthesis inhibitors,antidepressants, anxiolytic drugs, antipsychotic drugs, anti-amyotrophiclateral sclerosis drugs, anti-Huntington's drugs, anti-Alzheimer'sdrugs, anti-epileptic drugs, and/or steroids.

Yet another aspect of the present disclosure provides for a method oftreating a mitochondria-associated disease, disorder, or condition in asubject, the method comprising administering to the subject atherapeutically effective amount of a mitofusin activator.

In some aspects, the subject may be diagnosed with or may be suspectedof having a mitochondria-associated disease, disorder, or condition.

In some aspects, the mitochondria-associated disease, disorder, orcondition may be one or more of: a central nervous system (CNS) orperipheral nervous system (PNS) injury or trauma, such as trauma to theCNS or PNS, crush injury, spinal cord injury (SCI), traumatic braininjury, stroke, optic nerve injury, or related conditions that involveaxonal disconnection; a chronic neurodegenerative condition whereinmitochondrial fusion, fitness, or trafficking are impaired; a disease ordisorder associated with mitofusin 1 (MFN1) or mitofusin 2 (MFN2) ormitochondrial dysfunction, fragmentation, or fusion; dysfunction in MFN1or MFN2 unfolding; mitochondria dysfunction caused by mutations; adegenerative neurological condition, such as Alzheimer's disease,Parkinson's disease, Charcot-Marie-Tooth disease, or Huntington'sdisease; hereditary motor and sensory neuropathy, autism, autosomaldominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease,cancer, mitochondrial myopathy, diabetes mellitus and deafness (DAD),Leber's hereditary optic neuropathy (LHON), Leigh syndrome, subacutesclerosing encephalopathy, neuropathy, ataxia, retinitis pigmentosa, andptosis (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE),myoclonic epilepsy with ragged red fibers (MERRF), mitochondrialmyopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms(MELAS), mtDNA depletion, mitochondrial neurogastrointestinalencephalomyopathy (MNGIE), dysautonomic mitochondrial myopathy,mitochondrial channelopathy, and/or pyruvate dehydrogenase complexdeficiency (PDCD/PDH).

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A illustrates a structural model of human MFN2 in theclosed/inactive (left) and open/active conformation (right) that may bepromoted by mitofusin activators. FIG. 1B depicts a pharmacophoremodeling of interacting amino acid side chains that resulted in theprototype mitofusin small molecule agonist (Rocha, et al.; Science2018).

FIGS. 2A-2C depict structure-function relationships of MiM 111 cis- andtrans-isostereomers (see e.g., Example 3). FIG. 2A shows thedose-response curves for cis- (Cpd 15A) and trans-(Cpd 15B) MiM 111stereoisomers in cells expressing only MFN2. FIG. 2B shows thedose-response curves for cis-(Cpd 15A) and trans-(Cpd 15B) MiM 111stereoisomers in cells expressing only MFN1. “Cpd 2” represents ChimeraC, a prototype mitofusin activator for comparison. FIG. 2C shows theFRET analysis of conformational switching provoked by mitofusinactivators. Open conformation is more active. “Peptide” is MP1 mitofusinagonist peptide described in reference Franco Nature 2016 and depictedin FIG. 1A.

FIGS. 3A-3B depict chemical structures and corresponding NMR spectra ofMiM 111 cis- (FIG. 3A) and trans-(FIG. 3B) isostereomers (see e.g.,Example 3). Black arrows and arrowhead are specific to cis-isomer; greyarrowheads signify trans-isomer.

FIGS. 4A-4B depict in vivo (mouse) pharmacokinetic properties oftrans-MiM 111 (Cpd 15B) (see e.g., Example 5). FIG. 4A shows the totalplasma and brain concentrations after single dose IV injections. FIG. 4Bshows the steady state elimination kinetics after 3 days of continuoussubcutaneous infusion.

FIGS. 5A-5B shows the oral bioavailability and in vivo target engagementof trans-MiM 111 (Cpd 15B) (see e.g., Example 6). FIG. 5A shows theplasma levels after single dose IV (closed circles) or oral (opencircles) administration. FIG. 5B shows kymographs demonstratingmitochondrial motility in sciatic nerves of CMT2A mice 6 hours afteroral administration as in A. Motile mitochondria exhibit horizontaldisplacement (corresponding group quantitative data are on the right).

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery thatpharmacophore modeling of function-critical MFN2-derived interactingpeptides may produce structurally diverse small molecule peptidomimeticactivators useful to treat mitochondrial-associated diseases, disorders,and conditions. As shown herein, the present disclosure providesstereoisomer-specific chemicals or compositions for regulatingmitochondrial function. These compositions may be useful to correctorganelle, cell, and organ dysfunction caused by primary or secondarymitochondrial abnormalities that cause or contribute to diseasepathology and dysfunction.

Mitofusin Activators

The present disclosure provides for a class of stereoisomer-specifictrans-4-hydroxycyclohexyl derivative small molecules that promote achange in configuration of MFN1 and MFN2 leading to heightenedactivation. As described herein, a composition for the treatment of amitochondria-associated disease, disorder, or condition may comprise anactive trans-4-hydroxycyclohexyl mitofusin activator, such as apeptidomimetic (e.g., a small-molecule that mimics thechemico-structural features of a peptide). A peptidomimetic may be achemical peptidomimetic. For example, the peptidomimetic may mimic amitofusin-derived mini-peptide. Certain metabolites of the mitofusinactivator may also maintain activity in vivo.

The present disclosure describes functional activity of small moleculepeptidomimetics requiring mimicry of the endogenous peptide conformationin 3-dimensional space. Diastereomers of the same chemical mitofusinactivator prefer different 3-dimensional structures (FIGS. 1A-1B). Thus,cis- and trans-4-hydroxycyclohexyl derivatives exhibit dramaticallydifferent abilities to physically engage with and functionally activatetheir mitofusin protein targets (FIG. 2A-2C).

As described herein, a new generation of trans-4-hydroxycyclohexylderivative peptidomimetic small molecules has been developed exhibitingstereoisomer-specific functional activity. These compounds activatemitochondrial fusion by directing MFN1 and MFN2 to differentconformational states. The prototype small molecule peptidomimetics totarget MFN1 or MFN2 (described in Rocha, et al.; Science, 2018) had poorpharmacokinetic characteristics, making them “undruggable.” Describedherein are active stereoisomers of a structurally distinct class ofsmall molecule mitofusin activators that activate mitochondrial fusionand subcellular transport, have favorable pharmacokinetic properties,and may be used to correct mitochondrial and cellular dysfunction.

Mitofusin Mini-Peptide

As described herein, a peptide mitofusin activator may be anMFN2-derived mini-peptide as described in Franco, et al.; Nature 2016.

MFN Activator (Fusion-Promoting) Peptidomimetic

As described herein, a peptidomimetic may be a MFN activator(fusion-promoting) peptidomimetic that competes with endogenous MFN1 orMFN2 HR1-HR2 peptide-peptide interactions as described in Franco, etal.; Nature 2016 and Rocha, et al.; Science 2018.

The prototype mitofusin activator, Chimera C according to the presentdisclosure, includes the following compound:

Mitofusin Activators: Structurally Distinct Small Molecules thatActivate MFN1 and/or MFN2

The small molecule mitofusin activators described herein are allostericmitofusin activators designed in part using the pharmacophore HR1-HR2peptide-peptide interaction model described in Rocha, et al.; Science2018, but which are structurally distinct and of separate chemicalclasses from those reported by Rocha. An activator is a substance thatpartially or fully activates the protein to which it binds.

The mitofusin activator may comprise one or more compounds representedby formula (I):

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof, wherein R¹ may be selected from the following moieties:

Optionally, R¹ in formula (I) may be independently substituted by one ormore of the following groups: acetamide, C₁₋₈ alkoxy, amino, azo, Br,C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O,phenyl, S, sulfoxide, sulfone, and/or thiophene and optionally furthersubstituted with acetamide, alkoxy, amino, azo, Br, C₁₋₃ alkyl,carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S,sulfoxide, sulfone, and/or thiophene and the alkyl, cycloalkyl,heteroaryl, heterocyclyl, indole, or phenyl may be optionally furthersubstituted with one or more of the following groups: acetamide, alkoxy,amino, azo, Br, C₁₋₈alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈-heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone and/or thiophene.

Optionally, the R¹ group in formula (I) may be independently substitutedwith one or more of the following groups: hydroxyl; C₁₋₁₀ alkyl hydroxyl(i.e., C₁-C₁₀ alkoxides); amine; C₁₋₁₀ carboxylic acid; C₁₋₁₀ carboxyl;straight chain or branched C₁₋₁₀ alkyl, optionally containingunsaturation; a C₂₋₈ cycloalkyl optionally containing unsaturation orone oxygen or nitrogen atom; straight chain or branched C₁₋₁₀ alkylamine; heterocyclyl; heterocyclic amine; and/or aryl comprising phenyl,heteroaryl containing from one to four of the following heteroatoms: N,O, and/or S, unsubstituted phenyl ring, substituted phenyl ring,unsubstituted heterocyclyl, and substituted heterocyclyl. Optionally,the unsubstituted phenyl ring or substituted phenyl ring may beindependently substituted with one or more of the following groups:hydroxyl; C₁₋₁₀ alkyl hydroxyl (i.e., C₁-C₁₀ alkoxides); amine; C₁₋₁₀carboxylic acid; C₁₋₁₀ carboxyl; straight chain or branched C₁₋₁₀ alkyl,optionally containing unsaturation; straight chain or branched C₁₋₁₀alkyl amine, optionally containing unsaturation; a C₂₋₁₀ cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;straight chain or branched C₁₋₁₀ alkyl amine; heterocyclyl; heterocyclicamine; and/or aryl comprising phenyl and heteroaryl containing from oneto four of the following heteroatoms: N, O, and/or S. Optionally, theunsubstituted heterocyclyl or substituted heterocyclyl may beindependently substituted with one or more of the following groups:hydroxyl; C₁₋₁₀alkyl hydroxyl (i.e., C₁-C₁₀ alkoxides); amine; C₁₋₁₀carboxylic acid; C₁₋₁₀ carboxyl; straight chain or branched C₁₋₁₀ alkyloptionally containing unsaturation; straight chain or branched C₁₋₁₀alkyl amine optionally containing unsaturation; a C₂₋₈ cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;heterocyclyl; straight chain or branched C₁₋₁₀ alkyl amine; heterocyclicamine; and/or aryl comprising a phenyl and a heteroaryl containing fromone to four of the following heteroatoms: N, O, and S. Any of the abovemay be further optionally substituted.

In some aspects, R¹ in formula (I) may be optionally substituted by oneor more of the following groups: acetamide, alkoxy, amino, azo, Br, C₁₋₈alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl,C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S,sulfoxide, sulfone, and/or thiophene; and optionally further substitutedwith one or more of the following groups: acetamide, alkoxy, amino, azo,Br, C₁₋₃ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O,phenyl, S, sulfoxide, sulfone, or thiophene; wherein the alkyl,cycloalkyl, heteroaryl, heterocyclyl, indole, or phenyl, may beoptionally further substituted with one or more of acetamide, alkoxy,amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/or thiophene.

In another aspect of the disclosure, the mitofusin activator maycomprise one or more compounds represented by formula (II):

or a pharmaceutically acceptable salt thereof. In Formula (II), o may be0, 1, 2, 3, 4, or 5; p may be 0 or 1; and q may be 0, 1, 2, 3, 4, or 5with the proviso that the sum of o+p+q is not less than 3 or greaterthan 7; Z may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R²and R³ may be independently H, F, alkyl, or C₃₋₇ cycloalkyl; oroptionally, R² and R³ taken together may form a C₃₋₇ cycloalkyl orheterocycloalkyl; R⁴ and R⁵ may be independently H, F, alkyl, COR⁸, orC₃₋₇ cycloalkyl; or optionally, R⁴ and R⁵ taken together may form a C₃₋₇cycloalkyl or heterocycloalkyl; Y may be O, CR⁶R⁷, CR⁸═CR⁹, C≡C,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, NR⁸, S, SO₂, SONR⁹,NR⁹SO₂, NR⁸CO, CONR⁸, NR⁷CONR⁹; each R⁶ may be independently selectedfrom H, alkyl, and C₃₋₇ cycloalkyl; each R⁷ may be independentlyselected from H, alkyl, COR⁸ and C₃₋₇ cycloalkyl; or optionally, R⁶ andR⁷ taken together may form C₃₋₇ cycloalkyl; and each R⁸ may beindependently selected from H, alkyl, and C₃₋₇ cycloalkyl; each R⁹ maybe independently selected from H, alkyl, COR⁸ and C₃₋₇ cycloalkyl; oroptionally, R⁸ and R⁹ may be taken together to form C₃₋₇ cycloalkyl.

In some aspects, in the mitofusin activator of formula (II), o may be 0,1, 2, 3, 4, or 5; p may be 0 or 1; and q may be 0, 1, 2, 3, 4, or 5 withthe proviso that the sum of o+p+q is not less than 3 or greater than 7;Z may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Y may be O,CR⁶R⁷, cycloalkyl, or aryl; and R², R³, R⁴, R⁵, R⁶ and R⁷ may beindependently selected from H or alkyl.

In some aspects, in the mitofusin activator of formula (II), o may be 0,1, 2, 3, 4, or 5; p may be 0 or 1; and q may be 0, 1, 2, 3, 4, or 5 withthe proviso that the sum of o+p+q is not less than 3 or greater than 5;Z may be aryl or heteroaryl; Y may be O, CH₂, or cycloalkyl; R², R³, R⁴and R⁵ may each be H.

In some aspects, in the mitofusin activator of formula (II), o may be 0,1, 2, or 3; p may be 1; and q may be 0, 1, 2, or 3 with the proviso thatthe sum of o+p+q is not less than 3 or greater than 5; Z may be aryl orheteroaryl; Y may be cyclopropyl or cyclobutyl; R², R³, R⁴ and R⁵ mayeach be H.

In some aspects, in the mitofusin activator of formula (II), Z may bearyl or heteroaryl; Y may be 0 or CH₂; R², R³, R⁴ and R⁵ may each be H;o may be 0, 1, 2, 3, or 4; p may be 1; and q may be 0, 1, 2, 3, or 4with the proviso that the sum of o+p+q is 5.

In some aspects, in the mitofusin activator of formula (II), Z may bephenyl or heteroaryl, wherein the heteroaryl may contain from one tofour heteroatoms independently selected from N, O, and S, and whereinthe phenyl or heteroaryl may be substituted with zero to four of thefollowing independently-chosen substituents: R⁸, OR⁸, Cl, F, CN, CF₃,NR⁸R⁹, SO₂NR⁸R⁹, NR⁸SO₂R¹⁰, SO₂R⁹, CONR⁸R¹⁰, NR⁸COR¹⁰, C₃₋₇ cycloalkyl,and/or heterocycloalkyl; each R⁸ may be independently selected from H,alkyl, and C₃₋₇ cycloalkyl; each R⁹ may be independently selected fromH, alkyl, COR⁷ and C₃₋₇ cycloalkyl; or optionally, R⁸ and R⁹ takentogether may form a C₃₋₇ cycloalkyl; Y may be O or CH₂; R², R³, R⁴, andR⁵ may each be H; o may be 0, 1, 2, 3, or 4; p may be 1; and q may be 0,1, 2, 3, or 4 with the proviso that the sum of o+p+q is 5.

In some aspects, in the mitofusin activator of formula (II), Z may bephenyl or heteroaryl, wherein the heteroaryl may contain one to three ofthe following heteroatoms: N, O, and S, and wherein the phenyl orheteroaryl may contain zero to three of the following substituentsindependently selected from R⁸, OR⁸, Cl, F, CN, CF₃, NR⁸R⁹, SO₂R⁹,CONR⁸R¹⁰, NR⁸COR¹⁰, C₃₋₇ cycloalkyl, and/or heterocycloalkyl; Y may be Oor CH₂; R², R³, R⁴, and R⁵ may each be H; each R⁸ may independently beselected from H, alkyl, and C₃₋₇ cycloalkyl; each R⁹ may beindependently selected from H, alkyl, COR⁷ and C₃₋₇ cycloalkyl; oroptionally, R⁸ and R⁹ taken together may form a C₃₋₇ cycloalkyl; each R⁹may be alkyl or C₃₋₇ cycloalkyl; o may be 0, 1, 2, 3, or 4; p may be 1;and q may be 0, 1, 2, 3, or 4 with the proviso that the sum of o+p+q is5.

In some aspects, in the mitofusin activator of formula (II), Z may bephenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 6-pyrimidinyl,5-pyrimidinyl, 4-pyrimidinyl, or 2-pyrimidinyl, wherein the phenyl,pyridinyl, and pyrimidinyl moieties may independently contain zero totwo of the following independently-chosen substituents: R⁸, OR⁸, Cl, F,CN, CF₃, NR⁸R⁹, SO₂R⁹, CONR⁸R⁹, and/or NR⁸COR¹⁰; Y may be O or CH₂; R²,R³, R⁴, and R⁵ may each be H; each R⁸ may independently be H, alkyl, andC₃₋₇ cycloalkyl; each R⁹ may be independently selected from H, alkyl,COR⁷ and C₃₋₇ cycloalkyl; or optionally, R⁸ and R⁹ taken together mayform a C₃₋₇ cycloalkyl; R¹⁰ may be alkyl or C₃₋₇ cycloalkyl; o may be 0,1, 2, 3, or 4; p may be 1; and q may be 0, 1, 2, 3, or 4 with theproviso that the sum of o+p+q is 5.

Accordingly, in some aspects, in the mitofusin activator of formula(II), Z may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R² andR³ may be independently selected from H, F, alkyl, and C₃₋₇ cycloalkyl,or R² and R³ taken together may form a C₃₋₇ cycloalkyl orheterocycloalkyl; R⁴ and R⁵ may be independently selected from H, F,alkyl, and C₃₋₇ cycloalkyl, or R⁴ and R⁵ taken together may form a C₃₋₇cycloalkyl or heterocycloalkyl; Y is O, CR⁶R⁷; CR⁸CR⁹, C≡C, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, NR⁸, S, SO₂, SONR⁹, NR⁹SO₂, NR⁸CO,CONR⁸, or NR⁸CONR⁹; R⁶ may be H, F, alkyl or cycloalkyl; R⁷ may be H, F,alkyl or cycloalkyl; or R⁶ and R⁷ taken together may form a C₃₋₇cycloalkyl or heterocycloalkyl; R⁸ may be H, F, alkyl or cycloalkyl; R⁹may be H, F, alkyl or cycloalkyl; or R⁸ and R⁹ taken together may form aC₃₋₇ cycloalkyl; o is 0, 1, 2, 3, 4, or 5; p is 0 or 1; and q is 0, 1,2, 3, 4, or 5, provided that if Y is cycloalkyl, particularlycyclopropyl or cyclobutyl, the sum of o+p+q is not less than 3 orgreater than 5, and otherwise the sum of o+p+q is 5.

In particular aspects, in the mitofusin activator of formula (II), Z maybe aryl or heteroaryl; Y may be O, CR⁶R⁷, or cycloalkyl; R², R³, R⁴, R⁵,R⁶, and R⁷ may be H or alkyl; and p is 1. In other particular aspects, Ymay be cyclopropyl or cyclobutyl, or Y may be O or CH₂; R², R³, R⁴, andR⁵ may each be H; and p is 1. In still other particular aspects, Z maybe phenyl or heteroaryl, wherein the heteroaryl is 2-pyridinyl,3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,or 6-pyrimidinyl, wherein the phenyl or heteroaryl may be optionallysubstituted. Optional substitutions include 0 to 4, or 0 to 3, or 0 to 2substituents independently selected from R⁸, OR⁸, Cl, F, CN, CF₃, NR₈R₉,SO₂NR₈R₉, NR₈SO₂R₉, SO₂R₉, CONR₈R₉, NR₈COR₁₀, C₃₋₇ cycloalkyl, andheterocycloalkyl.

Mitofusin activators of the present disclosure may stimulatemitochondrial fusion, increase mitochondrial fitness, and enhancemitochondrial subcellular transport. More particular examples ofmitofusin activators suitable for achieving one or more of these resultsmay include trans-stereoisomers of 6-phenylhexanamide derivatives or apharmaceutically acceptable salt thereof, particularly 6-phenylhexanamide derivatives that are N-substituted with a trans-4-cyclohexylgroup. Compositions of the present disclosure and methods employingcomprising trans-stereoisomers of 6-phenylhexanamide derivatives maycontain greater than a 1:1 molar ratio of the trans-stereoisomerrelative to the cis-stereoisomer, such as at least about 60% or greatertrans-stereoisomer on a molar basis, or about 70% or greater, or about80% or greater, or about 90% or greater, or about 95% or greater, orabout 97% or greater, or about 99% or greater. Compositions may evencomprise trans-stereoisomer 6-phenylhexanamide derivatives that areracemically pure.

Particular examples of trans-stereoisomer 6-phenylhexanamide derivativeshaving activity for promoting mitofusin activation include, for example,

In another aspect of the present disclosure, a method of treating adisease for which a mitofusin activator is indicated may compriseadministering to a mammal in need thereof, such as a human, atherapeutically effective amount of a compound of formula (II)

or a pharmaceutically acceptable salt thereof. In Formula (II), o may be0, 1, 2, 3, 4, or 5; p may be 0 or 1; and q may be 0, 1, 2, 3, 4, or 5with the proviso that the sum of o+p+q is not less than 3 or greaterthan 7; Z may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R²and R³ may be independently H, F, alkyl, or C₃₋₇ cycloalkyl; oroptionally, R² and R³ taken together may form a C₃₋₇ cycloalkyl orheterocycloalkyl; R⁴ and R⁵ may be independently H, F, alkyl, COR⁸, orC₃₋₇ cycloalkyl; or optionally, R⁴ and R⁵ taken together may form a C₃₋₇cycloalkyl or heterocycloalkyl; Y may be O, CR⁶R⁷, CR⁸═CR⁹, C≡C,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, NR⁸, S, SO₂, SONR⁹,NR⁹SO₂, NR⁸CO, CONR⁸, NR⁷CONR⁹; each R⁶ may be independently selectedfrom H, alkyl, and C₃₋₇ cycloalkyl; each R⁷ may be independentlyselected from H, alkyl, COR⁸ and C₃₋₇ cycloalkyl; or optionally, R⁶ andR⁷ taken together may form C₃₋₇ cycloalkyl; and each R⁸ may beindependently selected from H, alkyl, and C₃₋₇ cycloalkyl; each R⁹ maybe independently selected from H, alkyl, COR⁸ and C₃₋₇ cycloalkyl; oroptionally, R⁸ and R⁹ may be taken together to form C₃₋₇ cycloalkyl. Anyof the mitofusin activators specified above may be employed for treatinga human or other mammal having or suspected of having amitochondria-associated disease, disorder or condition.

The mitochondria-associated disease, disorder or condition may be apheripheral nervous system (PNS) or central nervous system (CNS) geneticor non-genetic disorder, physical damage, and/or chemical injury. Insome aspects, in the method of treating a disease for which a mitofusinactivator is indicated, the PNS or CNS disorder may be selected from anyone or a combination of: a chronic neurodegenerative condition whereinmitochondrial fusion, fitness, or trafficking are impaired; a disease ordisorder associated with mitofusin-1 (MFN1) or mitofusin-2 (MFN2)dysfunction; a disease associated with mitochondrial fragmentation,dysfunction, or dysmotility; a degenerative neuromuscular condition suchas Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis (ALS),Huntington's disease, Alzheimer's disease, Parkinson's disease,hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH),diabetic neuropathy, chemotherapy-induced peripheral neuropathy, crushinjury, SCI, traumatic brain injury (TBI), stroke, optic nerve injury,and/or related conditions that involve axonal disconnection.

In some aspects of the disclosure, in the method of treating a diseasefor which a mitofusin activator is indicated, the composition mayfurther comprise a pharmaceutically acceptable excipient.

In some aspects of the disclosure, in the method of treating a CNSand/or PNS genetic and/or non-genetic neurodegenerative condition,injury, damage, and/or trauma comprising administering to the subject atherapeutically effective amount of a mitofusin activator according tothe present disclosure.

In some aspects of the disclosure, in the method of treating a CNS orPNS genetic or non-genetic neurodegenerative condition, injury, damage,or trauma, the subject may be diagnosed with or may be suspected ofhaving one or more of the following: a chronic neurodegenerativecondition wherein mitochondrial fusion, fitness, or trafficking areimpaired; a disease or disorder associated with MFN1 or MFN2dysfunction; a disease associated with mitochondrial fragmentation,dysfunction, or dysmotility; a degenerative neuromuscular condition(such as Charcot-Marie-Tooth disease, ALS, Huntington's disease,Alzheimer's disease, Parkinson's disease); hereditary motor and sensoryneuropathy, autism, ADOA, muscular dystrophy, Lou Gehrig's disease,cancer, mitochondrial myopathy, DAD, LHON, Leigh syndrome, subacutesclerosing encephalopathy, NARP, MNGIE, MERRF, MELAS, mtDNA depletion,MNGIE, dysautonomic mitochondrial myopathy, mitochondrial Channelopathy,PDCD/PDH, diabetic neuropathy, chemotherapy-induced peripheralneuropathy, crush injury, SCI, TBI, stroke, optic nerve injury, and/orrelated conditions that involve axonal disconnection.

The terms “imine” or “imino,” as used herein, unless otherwiseindicated, include a functional group or chemical compound containing acarbon-nitrogen double bond. The expression “imino compound,” as usedherein, unless otherwise indicated, refers to a compound that includesan “imine” or an “imino” group as defined herein. The “imine” or “imino”group may be optionally substituted.

The term “hydroxy,” as used herein, unless otherwise indicated, includes—OH. The “hydroxy” may be optionally substituted (e.g., incorporated inan alkoxide, phenoxide, or carboxylic acid ester).

The terms “halogen” and “halo”, as used herein, unless otherwiseindicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine,bromo, Br; and iodine, iodo, or I.

The term “acetamide,” as used herein, is an organic compound with theformula CH₃CONH₂. The “acetamide” may be optionally substituted.

The term “aryl,” as used herein, unless otherwise indicated, includes acarbocyclic aromatic group. Examples of aryl groups include, but are notlimited to, phenyl, benzyl, naphthyl, and anthracenyl. The “aryl” may beoptionally substituted.

The terms “amine” and “amino”, as used herein, unless otherwiseindicated, include a functional group that contains a nitrogen atom witha lone pair of electrons and wherein one or more hydrogen atoms havebeen replaced by a substituent such as, but not limited to, an alkylgroup or an aryl group. The “amine” or “amino” group may be optionallysubstituted.

The term “alkyl,” as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight or branchedmoieties, such as but not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, and octyl groups. Representative straight-chain loweralkyl groups include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl. Branched lower alkylgroups include, but are not limited to, isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 1-hexyl,2-hexyl, 3-hexyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl,2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl,2-methylheptyl, and 3-methylheptyl. Unsaturated alkyl groups may bereferred to as alkenyl (at least one carbon-carbon double bond) oralkynyl (at least one carbon-carbon triple bond) groups, which mayinclude, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, acetylenyl, propynyl,-1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, or 3-methyl-1 butynyl. Eand Z isomers may be present in any alkenyl group. The “alkyl,”“alkenyl,” or “alkynyl” may be optionally substituted.

The term “carboxyl,” as used herein, unless otherwise indicated,includes a functional group containing a carbon atom double bonded to anoxygen atom and single bonded to a hydroxyl group (—COOH). The“carboxyl” may be optionally substituted.

The term “acyl,” as used herein, unless otherwise indicated, includes afunctional group derived from an aliphatic carboxylic acid by removal ofthe hydroxyl (—OH) group. The “acyl” may be optionally substituted.

The term “alkoxy,” as used herein, unless otherwise indicated, includesO-alkyl groups wherein alkyl is as defined above and O representsoxygen. Representative alkoxy groups include, but are not limited to,—O-methyl, —O-ethyl, —O-n-propyl, —O-n-butyl, —O-n-pentyl, —O-n-hexyl,—O-n-heptyl, —O-n-octyl, —O-isopropyl, —O-sec-butyl, —O-isobutyl,—O-tert-butyl, —O-isopentyl, —O-2-methylbutyl, —O-2-methylpentyl,—O-3-methylpentyl, —O-2,2-dimethylbutyl, —O-2,3-dimethylbutyl,—O-2,2-dimethylpentyl, —O-2,3-dimethylpentyl, —O-3,3-dimethylpentyl,—O-2,3,4-trimethylpentyl, —O-3-methylhexyl, —O-2,2-dimethylhexyl,—O-2,4-dimethylhexyl, —O-2,5-dimethylhexyl, —O-3,5-dimethylhexyl,—O-2,4dimethylpentyl, —O-2-methylheptyl, —O-3-methylheptyl, —O-vinyl,—O-allyl, —O-1-butenyl, —O-2-butenyl, —O-isobutylenyl, —O-1-pentenyl,—O-2-pentenyl, —O-3-methyl-1-butenyl, —O-2-methyl-2-butenyl,—O-2,3-dimethyl-2-butenyl, —O-1-hexyl, —O-2-hexyl, —O-3-hexyl,—O-acetylenyl, —O-propynyl, —O-1-butynyl, —O-2-butynyl, —O-1-pentynyl,—O-2-pentynyl, —O-3-methyl-1-butynyl, —O-cyclopropyl, —O-cyclobutyl, —O—cyclopentyl, —O-cyclohexyl, —O-cycloheptyl, —O-cyclooctyl,—O-cyclononyl, —O— cyclodecyl, —O—CH₂-cyclopropyl, —O—CH₂-cyclobutyl,—O—CH₂-cyclopentyl, —O—CH₂-cyclohexyl, —O—CH₂-cycloheptyl,—O—CH₂-cyclooctyl, —O—CH₂-cyclononyl, —O—CH₂-cyclodecyl,—O—(CH₂)_(n)-cyclopropyl, —O—(CH₂)_(n)-cyclobutyl,—O—(CH₂)_(n)-cyclopentyl, —O—(CH₂)_(n)-cyclohexyl,—O—(CH₂)_(n)-cycloheptyl, —O—(CH₂)_(n)-cyclooctyl,—O—(CH₂)_(n)-cyclononyl, and/or —O—(CH₂)_(n)-cyclodecyl. The alkoxy maybe saturated, partially saturated, or unsaturated. The “alkoxy” may beoptionally substituted. In any example above, n may be from one to abouttwenty.

The term “cycloalkyl,” as used herein, unless otherwise indicated,includes a non-aromatic, saturated, partially saturated, or unsaturated,monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbonreferred to herein containing a total of from 3 to 10 carbon atoms.Examples of cycloalkyls include, but are not limited to, C₃₋₁₀cycloalkyl groups including cyclopropyl, cyclobutyl, cyclopentyl,cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl,1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. The term“cycloalkyl” also includes lower alkyl-cycloalkyl, wherein lower alkyland cycloalkyl are as defined herein. Examples of -loweralkyl-cycloalkyl groups include, but are not limited to,—CH₂-cyclopropyl, —CH₂-cyclobutyl, —CH₂-cyclopentyl,—CH₂-cyclopentadienyl, —CH₂-cyclohexyl, —CH₂-cycloheptyl, and/or—CH₂-cyclooctyl. The “cycloalkyl” may be optionally substituted.

The term “heterocyclic” as used herein, unless otherwise indicated,includes an aromatic group or non-aromatic cycloalkyl group in which oneto four of the ring carbon atoms are independently replaced with one ormore of O, S, and N. Aromatic heterocyclic groups are referred to as“heteroaryl” groups. Non-aromatic heterocyclic groups are referred to as“heterocyclyl” groups. Representative examples of heterocyclic groupsinclude, but are not limited to, benzofuranyl, benzothiophene, indolyl,benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl,thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl,quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl,isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane,4,5-dihydro-1H-imidazolyl, and/or tetrazolyl. Heterocyclic groups may besubstituted or unsubstituted. Heterocyclic groups may also be bonded atany ring atom (i.e., at any carbon atom or heteroatom of theheterocyclic ring). The heterocyclic group may be saturated, partiallysaturated, or unsaturated.

The term “indole,” as used herein, is an aromatic heterocyclic organiccompound with formula C₈H₇N. It has a bicyclic structure containing asix-membered benzene ring fused to a five-membered nitrogen-containingpyrrole ring. The “indole” may be optionally substituted.

The term “cyano,” as used herein, unless otherwise indicated, includes a—CN group.

The term “alcohol,” as used herein, unless otherwise indicated, includesa compound in which a hydroxy functional group (—OH) is bound to acarbon atom. In particular, this carbon atom may be saturated, havingsingle bonds to three other atoms. The “alcohol” may be optionallysubstituted. The “alcohol” may be a primary, secondary, or tertiaryalcohol.

The term “solvate” is intended to mean a solvated form of a specifiedcompound that retains the effectiveness of such compound. Examples ofsolvates include compounds of the invention in combination with, but notlimited to, one or more of: water, isopropanol, ethanol, methanol,dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

The term “stereoisomer” encompasses both optical isomers, such asenantiomers or diastereomers, the latter existing due to more than onecenter of chirality in the molecule, as well as geometrical isomers(cis/trans isomers or diastereomers).

A composition comprising the trans-stereoisomer 6-phenylhexanamidederivative mitofusin activator or a pharmaceutically acceptable saltthereof of the disclosure may comprise the trans-stereoisomer in greateramount than the cis-stereoisomer. A method of using the compositioncomprising the trans-stereoisomer 6-phenylhexanamide derivativemitofusin activator or a pharmaceutically acceptable salt thereof of thedisclosure may be such that the composition comprises thetrans-stereoisomer in greater amount than the cis-stereoisomer.

The term “mmol,” as used herein, is intended to mean millimole. The term“equiv” and “eq.,” as used herein, are intended to mean equivalent. Theterm “mL,” as used herein, is intended to mean milliliter. The term “g,”as used herein, is intended to mean gram. The term “kg,” as used herein,is intended to mean kilogram. The term “μg,” as used herein, is intendedto mean micrograms. The term “h,” as used herein, is intended to meanhour. The term “min,” as used herein, is intended to mean minute. Theterm “M,” as used herein, is intended to mean molar. The term “μL,” asused herein, is intended to mean microliter. The term “μM,” as usedherein, is intended to mean micromolar. The term “nM,” as used herein,is intended to mean nanomolar. The term “N,” as used herein, is intendedto mean normal. The term “amu,” as used herein, is intended to meanatomic mass unit. The term “° C.,” as used herein, is intended to meandegree Celsius. The term “wt/wt,” as used herein, is intended to meanweight/weight. The term “v/v,” as used herein, is intended to meanvolume/volume. The term “MS,” as used herein, is intended to mean massspectrometry. The term “HPLC,” as used herein, is intended to mean highperformance liquid chromatography. The term “RT,” as used herein, isintended to mean room temperature or retention time, depending oncontext. The term “e.g.,” as used herein, is intended to mean forexample. The term “N/A,” as used herein, is intended to mean not testedor not applicable.

As used herein, the expression “pharmaceutically acceptable salt” refersto pharmaceutically acceptable organic or inorganic salts of a compoundof the invention. Suitable salts include, but are not limited, tosulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and/or pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate))salts. A pharmaceutically acceptable salt may involve the inclusion ofanother molecule such as an acetate ion, a succinate ion, or othercounterion. The counterion may be any organic or inorganic moiety thatstabilizes the charge on the parent compound. Furthermore, apharmaceutically acceptable salt may have more than one charged atom inits structure. Instances where multiple charged atoms are part of thepharmaceutically acceptable salt may have multiple counterions. Hence, apharmaceutically acceptable salt may have one or more charged atomsand/or one or more counterion. As used herein, the expression“pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and a compound of the invention. Examples ofsolvents that form pharmaceutically acceptable solvates include, but arenot limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and/or ethanolamine. As used herein, theexpression “pharmaceutically acceptable hydrate” refers to a compound ofthe invention, or a salt thereof, that further includes a stoichiometricor non-stoichiometric amount of water bound by non-covalentintermolecular forces.

Each of the states, diseases, disorders, and conditions, describedherein, as well as others, can benefit from compositions and methodsdescribed herein. Generally, treating a state, disease, disorder, orcondition includes preventing or delaying the appearance of clinicalsymptoms in a mammal that may be afflicted with or predisposed to thestate, disease, disorder, or condition but does not yet experience ordisplay clinical or subclinical symptoms thereof. Treating can alsoinclude inhibiting the state, disease, disorder, or condition, e.g.,arresting or reducing the development of the disease or at least oneclinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Mitofusin 1 and Mitofusin 2

Mitofusins (MFN) 1 and 2, named for their central roles in mitochondrialfusion, are attractive drug targets because their regulatory functioningin mitochondrial dynamics and quality/quantity control is perturbed inseveral neurodegenerative disorders. In particular, genetic mutationsthat abrogate or impair MFN2 functioning and therefore suppressmitochondrial fusion cause the rare autosomal dominant neurodegenerativecondition Charcot-Marie-Tooth disease type 2A (CMT2A), for which thereis currently no disease-altering therapy. Moreover, accumulatingexperimental evidence supports an important role for MFN1 and MFN2 inheart disease. A pharmacological means of enhancing mitofusin functionhas the potential to correct the underlying cause of CMT2A and othercardiac or neurodegenerative diseases caused by mitochondrialdys-dynamism. MFN1 and MFN2 form homo (MFN1-MFN1 or MFN2-MFN2) or hetero(MFN1-MFN2) trans-dimers between mitochondria. This process is referredto as mitochondrial tethering, and is the requisite first step inmitochondrial fusion essential for metabolic cellular health (Koshiba,T., et al.; Science 305:858-62, 2004). Mitochondrial tethering andfusion depend upon a shift in MFN protein conformation, from a moreclosed resting state to a more open active state. MFN conformation isregulated by intra-molecular peptide-peptide interactions (PPI) betweenalpha helices in the stalk region of the protein (Franco, A., et al.;Nature 540:74-79, 2016). In human (h)MFN2, phosphorylation of serine 378by mitochondrial PINK1 kinase promotes a tight alpha helix within theinteracting peptide, directing critical Va372, Met 376, and His380 aminoacid side chains to their interacting partners Leu727, Leu723, andLys720 respectively (Rocha, A. G., et al.; Science 360:336-41, 2018). Astrong PPI promotes a folded protein conformation that decreases theprobability of trans MFN-MFN dimer formation between mitochondria (e.g.,is unfavorable for mitochondrial tethering/fusion). When serine 378 isnot phosphorylated the interacting peptide alpha helix partiallyunwinds, weakening the PPI and increasing the probability that theprotein will unfold to permit MFN trans-dimerization and subsequentmitochondrial fusion (Rocha, A. G., et al.; Science 2018).

Franco, et al. (Franco, A., et al.; Nature 2016) described an 18 aminoacid peptide, modified slightly from hMFN2 amino acids 367-384, thatcompetitively inhibited intra-molecular PPI in MFN1 and MFN2, thuspromoting the protein conformation favoring mitochondrial fusion. Byunderstanding the critical interacting amino acids within this activatorpeptide, a pharmacophore model was developed leading to identificationof a prototype small molecule mitofusin activator, Chimera B-A/I, whichmimicked mitofusin activator peptide effects by promoting mitochondrialfusion after topical application to mitofusin deficient cultured cellsand by increasing mitochondrial motility in ex vivo CMT2A nerves (Rocha,A. G., et al.; Science 2018). Clinical application of small moleculemitofusin activators having drug-like properties promises the firstdisease-altering therapy for CMT2A and may open the door to a noveltherapeutic approach of enhancing mitochondrial fusion in multiplediseases with impaired mitochondrial dynamics.

Mitochondria-Associated Diseases, Disorders, or Conditions

The present disclosure provides for compositions and methods oftreatment for treating mitochondria-related diseases, disorders, orconditions, including diseases or disorders associated with MFN1 and/orMFN2 and mitochondrial dysfunction. A mitochondria-associated disease,disorder, or condition may be a disease primarily caused by orsecondarily associated with mitochondrial dysfunction, fragmentation, orloss-of-fusion, or associated with dysfunction in MFN1 or MFN2 catalyticactivity or conformational unfolding. Mitochondrial dysfunction may becaused by genetic mutations of mitofusins or other (nuclear ormitochondrial encoded) genes, or may be caused by physical, chemical, orenvironmental injury to the CNS or PNS.

Mitochondria transit within cells and undergo fusion to exchange genomesand promote mutual repair. Mitochondrial fusion and subcellulartrafficking are mediated in part by MFN1 and MFN2. Genetic mutations inMFN2 that suppress mitochondrial fusion and motility causeCharcot-Marie-Tooth Disease, type 2A (CMT2A), the most common heritableaxonal neuropathy. Mitochondrial fragmentation, dysfunction, anddysmotility are also central features of other genetic neurodegenerativesyndromes, such as amyotrophic lateral sclerosis, Huntington's disease,Parkinson's disease, and Alzheimer's disease. Because no therapeuticsexist that directly enhance mitochondrial fusion or trafficking, thesediseases are unrelenting and considered irreversible.

Examples of mitochondria-associated diseases, disorders, and conditionsinclude, but are not limited to, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Charcot-Marie-Tooth Disease (type 2A)(CMT), hereditary motor and sensory neuropathy, autism, ADOA, musculardystrophy, Lou Gehrig's disease, cancer, mitochondrial myopathy, DAD,LHON, Leigh syndrome, subacute sclerosing encephalopathy, NARP, MNGIE,MERRF, MELAS, mtDNA depletion, MNGIE, dysautonomic mitochondrialmyopathy, mitochondrial channelopathy, and/or PDCD/PDH.

Symptoms that may be treated with the methods as described hereininclude, but are not limited to, poor growth, loss of musclecoordination, muscle paralysis and atrophy, visual problems, hearingproblems, learning disabilities, heart disease, liver disease, kidneydisease, gastrointestinal disorders, respiratory disorders, neurologicalproblems, autonomic dysfunction, and dementia.

Neurodegenerative Disease

As described herein, trans-stereoisomer 6-phenylhexanamide derivativemitofusin activators may rapidly reverse mitochondrial dysmotility insciatic nerve axons of a mouse model of Charcot-Marie-Tooth disease,type 2A. Because impaired mitochondrial fusion, fitness, and/ortrafficking also contribute to neuronal degeneration in variousneurodegenerative diseases (e.g., in Charcot-Marie-Tooth disease(CMT2A), Huntington's disease, Parkinson's disease, and Alzheimer'sdisease, and especially in ALS), the present disclosure provides forcompositions (e.g., compositions containing mitofusin activators) andmethods to treat such neurodegenerative diseases, disorders, and/orconditions.

Examples of neurodegenerative diseases, disorders and conditions includea disease of impaired neuronal mitochondrial dynamism or trafficking,such as, but not limited to, a hereditary motor and sensory neuropathy(HMSN) (e.g., CMT1 (a dominantly inherited, hypertrophic, predominantlydemyelinating form), CMT2 (a dominantly inherited predominantly axonalform), Dejerine-Sottas (severe form with onset in infancy), CMTX(inherited in an X-linked manner), and CMT4 (includes the variousdemyelinating autosomal recessive forms of Charcot-Marie-Tooth disease);hereditary sensory and autonomic neuropathy type IE, hereditary sensoryand autonomic neuropathy type II, hereditary sensory and autonomicneuropathy type V, HMSN types 1A and 1B (e.g., dominantly inheritedhypertrophic demyelinating neuropathies), HMSN type 2 (e.g., dominantlyinherited neuronal neuropathies), HMSN type 3 (e.g., hypertrophicneuropathy of infancy [Dejerine-Sottas]), HMSN type 4 (e.g.,hypertrophic neuropathy [Refsum] associated with phytanic acid excess),HMSN type 5 (associated with spastic paraplegia), and/or HMSN type 6(e.g., with optic atrophy)).

Other examples of neurodegenerative diseases, disorders, and conditionsinclude, but are not limited to, Alzheimer's disease, ALS, Alexanderdisease, Alpers' disease, Alpers-Huttenlocher syndrome,alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Artssyndrome, ataxia neuropathy spectrum, ataxia (e.g., with oculomotorapraxia, autosomal dominant cerebellar ataxia, deafness, andnarcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay,Batten disease, beta-propeller protein-associated neurodegeneration,cerebro-oculo-facio-skeletal syndrome (COFS), corticobasal degeneration,CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease,CLN6 disease, CLN7 disease, CLN8 disease, cognitive dysfunction,congenital insensitivity to pain with anhidrosis, dementia, familialencephalopathy with neuroserpin inclusion bodies, familial Britishdementia, familial Danish dementia, fatty acid hydroxylase-associatedneurodegeneration, Friedreich's Ataxia, Gerstmann-Straussler-ScheinkerDisease, GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., withretinitis pigmentosa), Huntington's disease, infantile neuroaxonaldystrophy, infantile-onset ascending hereditary spastic paralysis,infantile-onset spinocerebellar ataxia, juvenile primary lateralsclerosis, Kennedy's disease, Kuru, Leigh's Disease, Marinesco-Sjögrensyndrome, mild cognitive impairment (MCI), mitochondrial membraneprotein-associated neurodegeneration, motor neuron disease, monomelicamyotrophy, motor neuron diseases (MND), multiple system atrophy,multiple system atrophy with orthostatic hypotension (Shy-DragerSyndrome), multiple sclerosis, multiple system atrophy,neurodegeneration in down's syndrome (NDS), neurodegeneration of aging,neurodegeneration with brain iron accumulation, neuromyelitis optica,pantothenate kinase-associated neurodegeneration, opsoclonus myoclonus,prion disease, progressive multifocal leukoencephalopathy, Parkinson'sdisease, Parkinson's disease-related disorders, polycysticlipomembranous osteodysplasia with sclerosing leukoencephalopathy, priondisease, progressive external ophthalmoplegia, riboflavin transporterdeficiency neuronopathy, Sandhoff disease, spinal muscular atrophy(SMA), spinocerebellar ataxia (SCA), striatonigral degeneration,transmissible spongiform encephalopathies (prion diseases), and/orWallerian-like degeneration.

Charcot-Marie-Tooth (CMT) Disease Type 2A

Charcot-Marie-Tooth type 2A (CMT2A) disease is an example of anon-curable neurodegenerative disease/axonal neuropathy, disorder, orcondition caused by mutations of MFN2 and for which there are currentlyno disease-modifying treatments. As described herein, it was discoveredthat severely impaired mitochondrial transport from neuron cell body inthe spinal cord to distal neuronal synapse in the lower leg or hand (inaddition to smaller mitochondria size as is widely recognized) is acentral factor in CMT2A disease onset and progression. CMT2A is aprogressive neuromuscular disease that typically causes muscle weaknessand wasting in the distal legs/feet in children of ages 1-8 years, thenupper limbs, ultimately producing severe muscle wasting, skeletaldeformities, and permanent disability. The present disclosure providesfor the correction of impaired neuronal mitochondria transport as atherapeutic target in this disease. Data showed that administration of atrans-6-phenylhexanamide mitofusin activators promoted the mitochondriato move along neuronal axons in mouse models where mitochondria were notpreviously moving, and reversed disease-associated defects inneuromuscular function, which is applicable in any neuropathy (e.g.,Huntington's disease, ALS, ALS-like sclerosis, and/or Alzheimer'sdisease).

Neurological and Neurodegenerative Diseases

As described herein, trans-4-hydroxycyclohexyl 6-phenylhexanamidederivative mitofusin activators may rapidly reverse mitochondrialdysmotility in sciatic nerve axons of a mouse model ofCharcot-Marie-Tooth disease type 2A. It is currently believed thatimpaired mitochondrial trafficking also contribute to neuronaldegeneration in various neurological diseases (e.g., in Huntington'sdisease, Parkinson's disease, and Alzheimer's disease, and especially inALS). As such, the present disclosure provides for methods andcompositions to treat neurological diseases, disorders, or conditions.For example, a neurological disease, disorder, or condition may be, butis not limited to, abulia; agraphia; alcoholism; alexia; alien handsyndrome; Allan-Herndon-Dudley syndrome; alternating hemiplegia ofchildhood; Alzheimer's disease; amaurosis fugax; amnesia; ALS; aneurysm;angelman syndrome; anosognosia; aphasia; apraxia; arachnoiditis;Arnold-Chiari malformation; asomatognosia; Asperger syndrome; ataxia;attention deficit hyperactivity disorder; atr-16 syndrome; auditoryprocessing disorder; autism spectrum; Behcets disease; bipolar disorder;Bell's palsy; brachial plexus injury; brain damage; brain injury; braintumor; Brody myopathy; Canavan disease; capgras delusion; carpal tunnelsyndrome; causalgia; central pain syndrome; central pontinemyelinolysis; centronuclear myopathy; cephalic disorder; cerebralaneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); cerebraldysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK syndrome);cerebral gigantism; cerebral palsy; cerebral vasculitis; cervical spinalstenosis; Charcot-Marie-Tooth disease; chiari malformation; chorea;chronic fatigue syndrome; chronic inflammatory demyelinatingpolyneuropathy (CIDP); chronic pain; Cockayne syndrome; Coffin-Lowrysyndrome; coma; complex regional pain syndrome; compression neuropathy;congenital facial diplegia; corticobasal degeneration; cranialarteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulativetrauma disorders; Cushing's syndrome; cyclothymic disorder; cyclicvomiting syndrome (CVS); cytomegalic inclusion body disease (CIBD);cytomegalovirus infection; Dandy-Walker syndrome; dawson disease; deMorsier's syndrome; Dejerine-Klumpke palsy; Dejerine-Sottas disease;delayed sleep phase syndrome; dementia; dermatomyositis; developmentalcoordination disorder; diabetic neuropathy; diffuse sclerosis; diplopia;disorders of consciousness; down syndrome; Dravet syndrome; duchennemuscular dystrophy; dysarthria; dysautonomia; dyscalculia; dysgraphia;dyskinesia; dyslexia; dystonia; empty sella syndrome; encephalitis;encephalocele; encephalotrigeminal angiomatosis; encopresis; enuresis;epilepsy; epilepsy-intellectual disability in females; erb's palsy;erythromelalgia; essential tremor; exploding head syndrome; Fabry'sdisease; Fahr's syndrome; fainting; familial spastic paralysis; febrileseizures; Fisher syndrome; Friedreich's ataxia; fibromyalgia; Foville'ssyndrome; fetal alcohol syndrome; fragile x syndrome; fragilex-associated tremor/ataxia syndrome (FXTAS); Gaucher's disease;generalized epilepsy with febrile seizures plus; Gerstmann's syndrome;giant cell arteritis; giant cell inclusion disease; globoid cellleukodystrophy; gray matter heterotopia; Guillain-Barre syndrome;generalized anxiety disorder; HTLV-1 associated myelopathy;Hallervorden-Spatz syndrome; head injury; headache; hemifacial spasm;hereditary spastic paraplegia; heredopathia atactica polyneuritiformis;herpes zoster oticus; herpes zoster; Hirayama syndrome; Hirschsprung'sdisease; Holmes-Adie syndrome; holoprosencephaly; Huntington's disease;hydranencephaly; hydrocephalus; hypercortisolism; hypoxia;immune-mediated encephalomyelitis; inclusion body myositis;incontinentia pigmenti; infantile refsum disease; infantile spasms;inflammatory myopathy; intracranial cyst; intracranial hypertension;isodicentric 15; Joubert syndrome; Karak syndrome; Kearns-Sayresyndrome; Kinsbourne syndrome; Kleine-Levin syndrome; Klippel Feilsyndrome; Krabbe disease; Kufor-Rakeb syndrome; Lafora disease;Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateralmedullary (Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy;leukoencephalopathy with vanishing white matter; lewy body dementia;lissencephaly; locked-in syndrome; Lou Gehrig's disease (amyotrophiclateral sclerosis (ALS)); lumbar disc disease; lumbar spinal stenosis;lyme disease—neurological sequelae; Machado-Joseph disease(spinocerebellar ataxia type 3); macrencephaly; macropsia; mal dedebarquement; megalencephalic leukoencephalopathy with subcorticalcysts; megalencephaly; Melkersson-Rosenthal syndrome; menieres disease;meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly;micropsia; migraine; Miller Fisher syndrome; mini-stroke (transientischemic attack); misophonia; mitochondrial myopathy; mobius syndrome;monomelic amyotrophy; Morvan syndrome; motor neurone disease—see ALS;motor skills disorder; moyamoya disease; mucopolysaccharidoses;multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis;multiple system atrophy; muscular dystrophy; myalgic encephalomyelitis;myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonicencephalopathy of infants; myoclonus; myopathy; myotubular myopathy;myotonia congenita; narcolepsy; neuro-Behçet's disease;neurofibromatosis; neuroleptic malignant syndrome; neurologicalmanifestations of aids; neurological sequelae of lupus; neuromyotonia;neuronal ceroid lipofuscinosis; neuronal migration disorders;neuropathy; neurosis; Niemann-Pick disease; non-24-hour sleep-wakedisorder; nonverbal learning disorder; O'Sullivan-McLeod syndrome;occipital neuralgia; occult spinal dysraphism sequence; Ohtaharasyndrome; olivopontocerebellar atrophy; opsoclonus myoclonus syndrome;optic neuritis; orthostatic hypotension; otosclerosis; overuse syndrome;palinopsia; paresthesia; Parkinson's disease; paramyotonia congenita;paraneoplastic diseases; paroxysmal attacks; Parry-Romberg syndrome;pediatric autoimmune neuropsychiatric disorders associated withstreptococcoal infections (PANDAS); Pelizaeus-Merzbacher disease;periodic paralyses; peripheral neuropathy; pervasive developmentaldisorders; phantom limb/phantom pain; photic sneeze reflex; phytanicacid storage disease; Pick's disease; pinched nerve; pituitary tumors;pmg; polyneuropathy; polio; polymicrogyria; polymyositis; porencephaly;post-polio syndrome; postherpetic neuralgia (phn); postural hypotension;Prader-Willi syndrome; primary lateral sclerosis; prion diseases;progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive supranuclear palsy; prosopagnosia;pseudotumor cerebri; quadrantanopia; quadriplegia; rabies;radiculopathy; Ramsay Hunt syndrome type 1; Ramsay Hunt syndrome type 2;Ramsay Hunt syndrome type 3—see Ramsay-Hunt syndrome; Rasmussenencephalitis; reflex neurovascular dystrophy; refsum disease; REM sleepbehavior disorder; repetitive stress injury; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome;rhythmic movement disorder; Romberg syndrome; Saint Vitus' dance;Sandhoff disease; Schilder's disease (two distinct conditions);schizencephaly; sensory processing disorder; septo-optic dysplasia;shaken baby syndrome; shingles; Shy-Drager syndrome; Sjögren's syndrome;sleep apnea; sleeping sickness; snatiation; Sotos syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; spinal and bulbar muscular atrophy; spinocerebellar ataxia;split-brain; Steele-Richardson-Olszewski syndrome; stiff-personsyndrome; stroke; Sturge-Weber syndrome; stuttering; subacute sclerosingpanencephalitis; subcortical arteriosclerotic encephalopathy;superficial siderosis; Sydenham's chorea; syncope; synesthesia;syringomyelia; tarsal tunnel syndrome; tardive dyskinesia; tardivedysphrenia; Tarlov cyst; Tay-Sachs disease; temporal arteritis; temporallobe epilepsy; tetanus; tethered spinal cord syndrome; Thomsen disease;thoracic outlet syndrome; tic douloureux; Todd's Paralysis; tourettesyndrome; toxic encephalopathy; transient ischemic attack; transmissiblespongiform encephalopathies; transverse myelitis; traumatic braininjury; tremor; trichotillomania; trigeminal neuralgia; tropical spasticparaparesis; trypanosomiasis; tuberous sclerosis; 22q13 deletionsyndrome; Unverricht-Lundborg disease; vestibular schwannoma (acousticneuroma); Von Hippel-Lindau disease (VHL); viliuisk encephalomyelitis(VE); Wallenberg's syndrome; west syndrome; whiplash; Williams syndrome;Wilson's disease; y-linked hearing impairment; and/or Zellwegersyndrome.

Chemotherapy-Induced Peripheral Neuropathy (CIPN)

Cancer chemotherapy-induced sensory and motor neuropathies may beprevented or treated with the compostions and methods described herein.Although cancer continues to be a leading cause of mortality world-wide,early detection and improved cancer chemotherapeutics preferentiallyattacking rapidly dividing cells are favorably impacting this disease.Consequently, the number of cancer survivors is increasing andcollateral detrimental effects of successful cancer therapy are agrowing problem for cancer survivors. Chemotherapy-induced peripheralneuropathy is one of the most common complications of cancerchemotherapy, affecting 20% of all patients and almost 100% of patientsreceiving high doses of chemotherapeutic agents. Dose-dependentneurotoxicity of motor and sensory neurons can lead to chronic pain,hypersensitivity to hot, cold, and mechanical stimuli, and/or impairedneuromuscular control. The most common chemotherapeutic agents linked toCIPN are platinum, vinca alkaloids, taxanes, epothilones, and thetargeted proteasome inhibitor, bortezomib.

CIPN most commonly affects peripheral sensory neurons whose cell bodiesare located in dorsal root ganglia lacking the blood-brain barrier thatprotects other components of the central and peripheral nervous system.Unprotected dorsal root ganglion neurons are more sensitive to neuronalhyperexcitability and innate immune system activation evoked bycirculating cytotoxic chemotherapeutic agents. CIPN affects quality oflife, and is potentially disabling, because it provokes chronicneuropathic pain that, like other causes of neuralgia (e.g., postherpetic neuralgia, diabetic mononeuropathy), is refractory to analgesictherapy. Motor nerve involvement commonly manifests as loss of finemotor function with deterioration in hand writing, difficulty inbuttoning clothes or sewing, and sometimes upper and lower extremityweakness or loss of endurance. CIPN typically manifests within weeks ofchemotherapy and in many cases improves after chemotherapy treatmentends, although residual pain, sensory, or motor defects are observed inone third to one half of affected patients. Unfortunately, CIPN-limitedchemotherapy dosing can lead to delays, reduction, or interruption ofcancer treatment, thus shortening survival.

Mitochondrial dysfunction and oxidative stress are implicated in CIPNbecause of observed ultrastructural morphological abnormalities,impaired mitochondria DNA transcription and replication, induction ofmitochondrial apoptosis pathways, and reduction of experimental CIPNsigns by anticipatory mitochondrial protection. As described herein,trans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivators may enhance overall mitochondrial function in damagedneurons, increase mitochondrial transport to areas of neuronal damage,and accelerate in vitro neuron repair/regeneration afterchemotherapy-induced damage. For this reason, it is believed thattrans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivators may reduce neuronal injury conferred by chemotherapeuticagents in CIPN and accelerate regeneration/repair of nerves damaged bychemotherapeutic anticancer agents. Further testing of the CIPN damageprevention/repair and regeneration hypothesis will be further developedwith the trans-4-hydroxycyclohexyl 6-phenylhexanamide derivativemitofusin activators by evaluating their in vivo effectiveness. As such,the present disclosure provides for compositions and methods to treatcancer chemotherapy induced nerve injury and neuropathy.

CNS or PNS Injury or Trauma

Injury in the CNS or PNS (e.g., trauma to the CNS or PNS, crush injury,SCI, TBI, stroke, optic nerve injury, or related conditions that involveaxonal disconnection) may be treated with the compositions and methodsas described herein. The CNS includes the brain and the spinal cord andthe PNS is composed of cranial, spinal, and autonomic nerves thatconnect to the CNS.

Damage to the nervous system caused by mechanical, thermal, chemical, orischemic factors may impair various nervous system functions such asmemory, cognition, language, and voluntary movement. Most often, this isthrough accidental crush or transection of nerve tracts, or as anunintended consequence of medical interventions, that interrupt normalcommunications between nerve cell bodies and their targets. Other typesof injuries may include disruption of the interrelations between neuronsand their supporting cells or the destruction of the blood-brainbarrier.

As described herein, trans-4-hydroxycyclohexyl 6-phenylhexanamidederivative mitofusin activators may rapidly reverse mitochondrialdysmotility in neurons from mice or patients with various genetic orchemotherapeutic neurodegenerative diseases, in axons injured bychemotherapeutic agents, and in axons severed by physical injury. Forthis reason, it is believed that enhancing mitochondrial traffickingwith trans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivators may enhance regeneration/repair of physically damaged nerves,as in vehicular and sports injuries, penetration trauma from military orcriminal actions, and iatrogenic injury during invasive medicalprocedures. Further testing of the injury-regeneration hypothesis willbe further developed with the small molecule mitofusin activators forevaluation of their in vivo effectiveness. As such, the presentdisclosure provides for compositions and methods to treat physical nerveinjury.

As disclosed herein, mitochondrial motility is implicated in neuropathy.It is believed that mitochondrial motility is also implicated intraumatic crush or severance nerve injuries. After nerve laceration orcrush injury, nerves will either regenerate and restore neuromuscularfunction or fail to regenerate such that neuromuscular function inpermanently impaired. Trans-4-hydroxycyclohexyl 6-phenylhexanamidederivative mitofusin activators, as described herein, may increasemitochondrial trafficking, enabling the nerve to regenerate aftertraumatic injuries.

Formulation

The agents and compositions described herein may be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described previously (e.g., Remington'sPharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), which is incorporated herein by reference withrespect to its disclosure of pharmaceutically acceptable carriers). Suchformulations will contain a therapeutically effective amount of abiologically active agent described herein, which may be in purifiedform, together with a suitable amount of carrier to provide the form forproper administration to a subject.

The term “formulation” refers to a preparation of a drug in a formsuitable for administration to a subject such as a human or animal petor livestock. Thus, a “formulation” may include pharmaceuticallyacceptable excipients, including diluents or carriers.

The term “pharmaceutically acceptable,” as used herein, describessubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. One ofskill in the art will be familiar with suitable pharmaceuticallyacceptable substances. Examples of pharmaceutically acceptableingredients include those having monographs in United StatesPharmacopeia (USP 29) and National Formulary (NF 24), United StatesPharmacopeial Convention, Inc, Rockville, Md., 2005 (“USP/NF”), or amore recent edition, and the components listed in the continuouslyupdated Inactive Ingredient Search online database of the FDA. Otheruseful components that are not described in the USP/NF may also be used.

The term “pharmaceutically acceptable excipient,” as used herein,includes solvents, dispersion media, coatings, antibacterial agents,antifungal agents, isotonic, and absorption delaying agents. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art (see generally Remington's Pharmaceutical Sciences (A. R.Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar asany conventional media or agent is incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients may also be incorporated into thecompositions.

A “stable” formulation or composition refers to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

A formulation should suit the desired mode of administration. The agentsof use with the current disclosure may be formulated by known methodsfor administration to a subject using several routes including, but notlimited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations may also be used to affect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period. In order tomaintain a near-constant level of an agent in the body, the agent may bereleased from the dosage form at a rate that will replace the amount ofagent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers(e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules).

Agents or compositions described herein may also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Also provided herein is a process of treating a mitochondria-associateddisease, disorder, or condition in a subject in need of administrationof a therapeutically effective amount of a trans-4-hydroxycyclohexyl6-phenylhexanamide derivative mitofusin activator to prevent or treat amitochondria-associated disease, disorder, or condition.

For example, the compositions and methods described herein may be usedas a primary therapy for Charcot-Marie-Tooth or as an adjunctive therapyfor Huntington's disease, Parkinson's disease, Alzheimer's disease, orALS to retard or reverse disease progression.

As another example, the compositions and methods described herein may beused for the prevention or treatment of chemotherapy-induced peripheralneuropathy. For example as pre- and post-therapy for individualsundergoing scheduled chemotherapy for the treatment of cancer. Pre- andpost-chemotherapy treatment with trans-4-hydroxycyclohexyl6-phenylhexanamide derivative mitofusin activators may prevent,attenuate, and accelerate recovery from chemotherapy-induced peripheralneuropathy. This therapy may minimize sensory and motor neuronsusceptibility to chemotherapeutic agents and accelerate repair ofchemotherapy-induced neuronal damage by promoting mitochondrial fitnessand localization to areas of injury and regrowth.

As yet another example, the compositions and methods described hereinmay be used for the treatment of a physical injury. For example, as aprimary therapy for any contusive or laceration injury involving thespine or peripheral nerves (perhaps even the brain, (i.e., concussion),such as motor vehicle or sports injuries. This therapy may help restorenormal motor function by augmenting regeneration and repair of injuredneurons.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the therapeutic methods described hereinmay be a subject having, diagnosed with, suspected of having, or at riskfor developing a mitochondria-associated disease, disorder, orcondition. A determination of the need for treatment will typically beassessed by a history and physical exam consistent with the disease orcondition at issue. Diagnosis of the various conditions treatable by themethods described herein is within the skill of the art. The subject maybe an animal subject, including a mammal, such as horses, cows, dogs,cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, chickens,and humans. For example, the subject may be a human subject.

Generally, a safe and effective amount of a trans-4-hydroxycyclohexyl6-phenylhexanamide derivative mitofusin activator is, for example, thatamount that would cause the desired therapeutic effect in a subjectwhile minimizing undesired side effects. In various aspects, aneffective amount of a mitofusin activator described herein maysubstantially inhibit mitochondria-associated disease, disorder, orcondition, slow the progress of mitochondria-associated disease,disorder, or condition, or limit the development ofmitochondria-associated disease, disorder, or condition. For example, adesired therapeutic effect may be a delay in peripheral neuropathy(e.g., over the course of three years) compared to placebo assessed byslower increase in modified composite CMT neuropathy score. As anotherexample, a desired therapeutic effect may be reversal or absence ofprogression of peripheral neuropathy compared to placebo, as indicatedby lower or stable modified composite CMT neuropathy score. As yetanother example, a desired therapeutic effect may be reversal or absenceof progression of dysregulated motor function or increased regenerationand repair of injured neurons.

According to the methods described herein, administration may beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of a mitofusin activator may be employed in pure formor, where such forms exist, in pharmaceutically acceptable salt form andwith or without a pharmaceutically acceptable excipient. For example,the compounds of the present disclosure may be administered, at areasonable benefit/risk ratio applicable to any medical treatment, in asufficient amount to treat, reverse, prevent, or slow the progression ofmitochondria-associated disease, disorder, or condition.

The amount of a composition described herein that may be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein maybe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that may be expressed as the ratio LD₅₀/ED₅₀,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble, etal.; (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, may benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingmay also include inhibiting the state, disease, disorder, or condition(e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof). Furthermore, treating mayinclude relieving the disease (e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms). A benefit to a subject to be treated may beeither statistically significant or at least perceptible to the subjector to a physician.

Administration of a trans-4-hydroxycyclohexyl 6-phenylhexanamidederivative mitofusin activator may occur as a single event or over atime course of treatment. For example, a mitofusin activator may beadministered daily, weekly, bi-weekly, or monthly. For treatment ofacute conditions, the time course of treatment will usually be at leastseveral days. Certain conditions could extend treatment from severaldays to several weeks. For example, treatment could extend over oneweek, two weeks, or three weeks. For chronic conditions, treatment couldextend from several weeks to several months or even years.

Treatment in accord with the methods described herein may be performedprior to, concurrent with, or after conventional treatment modalitiesfor treating, preventing, or slowing the progression ofmitochondria-associated disease, disorder, or condition.

A trans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivator may be administered simultaneously or sequentially withanother agent, such as an antibiotic, an anti-inflammatory, or anotherneuroregenerative or neurotherapeutic agent. For example, atrans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivator may be administered simultaneously with another agent, such asan antibiotic or an anti-inflammatory. Simultaneous administration mayoccur through administration of separate compositions, each containingone or more of a mitofusin activator, an antibiotic, ananti-inflammatory, or another agent. Simultaneous administration mayoccur through administration of one composition containing two or moreof a mitofusin activator, an antibiotic, an anti-inflammatory, oranother agent. A trans-4-hydroxycyclohexyl 6-phenylhexanamide derivativemitofusin activator may be administered sequentially with an antibiotic,an anti-inflammatory, or another agent. For example, atrans-4-hydroxycyclohexyl 6-phenylhexanamide derivative mitofusinactivator may be administered before or after administration of anantibiotic, an anti-inflammatory, or another agent.

Administration

Agents and compositions described herein may be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition may be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

As discussed above, administration may be parenteral, pulmonary, oral,topical, transdermal (e.g., a transdermal patch) intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein may be administered in avariety of methods well known in the arts. Administration methods mayinclude, for example, methods involving oral ingestion, direct injection(e.g., systemic or stereotactic), implantation of cells engineered tosecrete the factor of interest, drug-releasing biomaterials, polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, implantable matrix devices, mini-osmoticpumps, implantable pumps, injectable gels and hydrogels, liposomes,micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm),microspheres (e.g., 1-100 μm), reservoir devices, a combination of anyof the above, or other suitable delivery vehicles to provide the desiredrelease profile in varying proportions. Other methods ofcontrolled-release delivery of agents or compositions will be known tothe skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump that may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition may beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system may be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents may be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery, tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to nontarget tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Kits

Also provided herein are kits. Such kits may include an agent orcomposition described herein and, in certain aspects, instructions foradministration. Such kits may facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition may be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to MFN1,MFN2, activator target peptides, or trans-4-hydroxycyclohexyl6-phenylhexanamide derivative mitofusin activators. Such packaging ofthe components separately can, if desired, be presented in a pack ordispenser device, which may contain one or more unit dosage formscontaining the composition. The pack may, for example, comprise metal orplastic foil such as a blister pack. Such packaging of the componentsseparately may also, in certain instances, permit long-term storagewithout losing activity of the components.

Kits may also include reagents in separate containers (e.g., sterilewater or saline) to be added to a lyophilized active component packagedseparately. For example, sealed glass ampules may contain a lyophilizedcomponent and in a separate ampule, sterile water, sterile saline orsterile each of which has been packaged under a neutral non-reactinggas, such as nitrogen. Ampules may consist of any suitable material,such as glass, organic polymers, such as polycarbonate, polystyrene,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include bottles that maybe fabricated from similar substances as ampules, and envelopes that mayconsist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, and thelike. Containers may have a sterile access port, such as a bottle havinga stopper that may be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to mix.Removable membranes may be glass, plastic, rubber, and the like.

In certain aspects, kits may be supplied with instructional materials.Instructions may be printed on paper or other substrate, and/or may besupplied as an electronic-readable medium, such as a floppy disc,mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and thelike. Detailed instructions may not be physically associated with thekit; instead, a user may be directed to an Internet web site specifiedby the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols may be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel, et al.; (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Metabolites

Metabolites of trans-(4-hydroxycyclohexyl)-6-phenylhexanamide wereisolated following in vivo administration and certain examples werefound to be highly active mitofusin activators. Among mitofusinactivators that are metabolites oftrans-(4-hydroxycyclohexyl)-6-phenylhexanamide include the followingcompounds:

Mitofusin activators having structures analogues to those producedmetabolically are also encompassed within the compounds disclosedherein. Any of the foregoing mitofusin activator metabolites may beadministered to a subject directly for purposes of managing amitochondria-associated disease, disorder or condition.

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some aspects, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain aspects of the present disclosure areto be understood as being modified in some instances by the term“about.” In some features, the term “about” is used to indicate that avalue includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some features, thenumerical parameters set forth in the written description and attachedclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by a particular feature. In someaspects, the numerical parameters should be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of some aspects of the present disclosureare approximations, the numerical values set forth in the specificexamples are reported as precisely as practicable. The numerical valuespresented in some aspects of the present disclosure may contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. The recitation of ranges of valuesherein is merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein.

In some aspects, the terms “a” and “an” and “the” and similar referencesused in the context of describing a particular aspect (especially in thecontext of certain of the following claims) may be construed to coverboth the singular and the plural, unless specifically noted otherwise.In some aspects, the term “or” as used herein, including the claims, isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” areopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and may cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand may cover other unlisted features.

All methods described herein may be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided with respect to certain aspects herein is intendedmerely to better illuminate the present disclosure and does not pose alimitation on the scope of the present disclosure otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements, embodiments, aspects, or features ofthe present disclosure disclosed herein are not to be construed aslimitations. Each group member may be referred to and claimedindividually or in any combination with other members of the group orother elements found herein. One or more members of a group may beincluded in, or deleted from, a group for reasons of convenience orpatentability. When any such inclusion or deletion occurs, thespecification is herein deemed to contain the group as modified thusfulfilling the written description of all Markush groups used in theappended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments, features, oraspects are possible without departing the scope of the presentdisclosure defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus may be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes may be made in the specific features that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the present disclosure.

Example 1. N-(cis-4-Hydroxycyclohexyl)-6-phenylhexanamide (MiM 111 Cis,Compound 15A)

To a solution of 6-phenylhexanoic acid 1.2 (200 mg, 1.04 mmol, 196 μL,1.00 eq.) and cis-4-hydroxycyclohexylamine 1.1 (174 mg, 1.14 mmol, 1.10eq.) and DIEA (269 mg, 2.08 mmol, 362 μL, 2.00 eq.) in DMF (3 mL) wasadded HOBt (169 mg, 1.25 mmol, 1.20 eq.) and EDCl (299 mg, 1.56 mmol,1.50 eq.). The mixture was stirred at 10° C. for 10 hours. The mixturewas diluted with water (20 mL) and extracted with EtOAc (10 mL×3). Theorganic phase was washed with 1M aqueous HCl (30 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by preparative HPLC. The title compound wasobtained as a light yellow gum. MS: m/z=290.1 (M+H)⁺; ¹H NMR (400 MHz):MeOD δ 7.25-7.22 (m, 2H), 7.17-7.11 (m, 3H), 3.84 (s, 1H), 3.70-3.65 (m,1H), 2.60 (t, J=7.6 Hz, 2H), 2.16 (t, J=7.2 Hz, 2H), 1.73-1.65 (m, 3H),1.65-1.59 (m, 9H), 1.36-1.34 (m, 2H). ¹³C NMR (400 MHz): MeOD δ:175.628, 143.895, 129.579, 129.414, 126.809, 66.945, 37.182, 36.885,32.558, 32.179, 29.863, 28.074, 27.168.

Example 2. N-(trans-4-Hydroxycyclohexyl)-6-phenylhexanamide (MiM 111Trans, Compound 15B)

To a solution of 6-phenylhexanoic acid 1.2 (200 mg, 1.04 mmol, 196 μL,1.00 eq.) and trans-4-hydroxycyclohexylamine 2.1 (174 mg, 1.14 mmol,1.10 eq.) and DIEA (269 mg, 2.08 mmol, 362 μL, 2.00 eq.) in DMF (3 mL)was added HOBt (169 mg, 1.25 mmol, 1.20 eq.) and EDCl (299 mg, 1.56mmol, 1.50 eq.). The mixture was stirred at 10° C. for 10 hours. Themixture was diluted with water (20 mL) and extracted with EtOAc (10mL×3). The organic phase was washed with 1M aqueous HCl (30 mL), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by preparative HPLC. The titlecompound was obtained as a white solid. MS: m/z=290.1 (M+H)⁺; ¹H NMR(400 MHz): MeOD δ 7.25-7.21 (m, 2H), 7.16-7.11 (m, 3H), 3.59-3.48 (m,1H), 2.60 (t, J=7.6 Hz, 2H), 2.13 (t, J=7.6 Hz, 2H), 2.11-1.92 (m, 2H),1.92-1.83 (m, 2H), 1.65-1.60 (m, 4H), 1.34-1.19 (m, 6H). ¹³C NMR (400MHz): MeOD δ: 175.660, 143.882, 129.590, 129.441, 126.819, 70.591,37.231, 36.864, 34.965, 32.519, 31.655, 29.804, 27.098.

Example 3: The Trans-Stereoisomer of MiM 111 is an Allosteric MitofusinAgonist, Whereas the Cis-Stereoisomer of MiM 111 is Inactive

Previously, chemical syntheses of candidate mitofusin activators sourcedstarting materials as a mixture of stereoisomers, resulting in productsthat were also mixtures of 2 or more isomers. It is recognized thattarget recognition and biological activity can differ betweenstereoisomers (Sundén, H., et al.; ChemMedChem 8:1283-94, 2013;Jafurulla, M., et al.; Biochim Biophys Acta 1838:158-63, 2014). However,due to the novelty of small molecule mitofusin activators as a drugclass, there are no data describing, nor even computer models that canpredict: 1. if stereoisomers affect mitofusin activator efficacy; and 2.if they do, which stereoisomer(s) have superior properties. Accordingly,cis- and trans-diastereomers ofN-(4-hydroxycyclohexyl)-6-phenylhexanamide, designated cis- andtrans-MiM 111, were individually synthesized, validated, andcharacterized (FIGS. 3A-3B). Remarkably, the cis form exhibited nodetectable mitofusin activating activity, measured as mitochondrialelongation, in either MFN1 or MFN2 deficient murine embryonicfibroblasts (FIGS. 2A-2B). By contrast, the trans form of MiM 111 wasequipotent to a prototype mitofusin agonist of the chemical classdescribed in Rocha, et al.; Science 2018, Chimera C. Functional activityof cis- and trans-N-(4-hydroxycyclohexyl)-6-phenylhexanamide mirroredtheir ability to provoke the characteristic change in MFN2 conformationthat underlies its activation (FIG. 2C), mechanistically linkingbiological effect and target engagement as a function of isomericstructure. The cis- and trans-stereoisomers of MiM 111 had similar invitro pharmacokinetic profiles (Table 1).

TABLE 1 In vitro functional and pharmacokinetic properties of MiM 111cis- and trans- diastereomers (Compounds 15A and 15B) EC₅₀ EC₅₀ (nM)(nM) t_(1/2) t_(1/2) PAMPA Mfn1 Mfn2 hPPB mPPB HLM MLM Pe Cpd StructureKO KO (%) (%) (min) (min) (nm/sec) 15A

inactive inactive 93.9 93.8 102 70 42.6 15B

7.7 9.5 90.4 96.7 >145 127 22.9

Example 4: Trans-Stereoisomers of MiM 111 Having Oxy-Substituted Linkersare Potent Mitofusin Activators with a Spectrum of Passive MembranePermeability Characteristics

The complete series of oxy-substituted linker analogs fortrans-N-(4-hydroxycyclohexyl)-6-phenylhexanamide were synthesized andevaluated for fusogenic activity and pharmacokinetic properties. All ofthe oxy-substituted trans-analogs retained excellent fusogenic activity,but the position of the oxygen within the linker altered the passivemembrane permeability characteristics of these compounds, with thecarbamate exhibiting 10-fold greater PAMPA, but reduced microsomalstability, compared to parenttrans-N-(4-hydroxycyclohexyl)-6-phenylhexanamide (Table 2).

TABLE 2 Functional and pharmacokinetic properties of oxy-substitutedlinker analogs of trans-MiM 111 ((Compound 15B) t_(1/2) t_(1/2) PAMPAEC₅₀ hPPB mPPB HLM MLM Pe Cpd Structure (nM) (%) (%) (min) (min)(nm/sec) 15B

5.54 90.4 96.7 >145 127 22.9 21

9.63 80.2 81.8 >145 >145 3.66 22

12.92 57.1 60.4 >145 >145 1.176 23

3.31 38.2 48.5 >145 >145 2.228 24

7 31 55.2 66.4 >145 >145 23.799 25

6.28 93.3 94.8 131.3 48.9 210.149

Compounds 21-25 were synthesized similarly to Compound 15B (Example 2),except incorporating an appropriate oxygenated 6-phenylhexanoic acidderivative for 6-phenylhexanoic acid 1.2. Namely, Compounds 21-25 weresynthesized by replacing 6-phenylhexanoic acid 1.2 with5-phenoxypentanoic acid, 4-(benzyloxy)butanoic acid,3-phenethoxypropanoic acid, 2-(3-phenylpropoxy)acetic acid, or4-phenylbutylcaronochloridate, respectively.

Compound 21: HPLC: RT: 2.17 min, purity: 99.9%. LC-MS: RT=0.830 min,m/z=292.3 (M+H)⁺. ¹H NMR: 400 MHz, MeOD. δ 7.25 (t, J=8.0 16.4 Hz, 2H),6.9 (d, J=8.4 Hz, 3H), 3.9 (m, 2H), 3.51-3.63 (m, 2H), 2.24 (s, 2H),1.92-1.97 (m, 4H), 1.77-1.70 (m, 4H), 1.30-1.37 (m, 4H). ¹³C NMR: (400MHz, MeOD) δ (176, 161, 131, 122, 116, 71.2, 69.2, 37.6, 35.6, 32.3,30.7, 24.5) ppm.

Compound 22: HPLC: RT: 1.80 min, purity: 99.8%. LC-MS: RT=0.802 min,m/z=292.3 (2M+Na)⁺. ¹H NMR: (400 MHz, MeOD) δ 7.25-7.33 (m, 5H), 4.48(s, 2H), 3.46-3.59 (m, 4H), 2.24 (t, J=7.2, 2H), 1.82-1.92 (m, 6H),1.20-1.33 (m, 4H). ¹³C NMR: (400 MHz, MeOD) δ (175, 139, 130, 129, 128,74.0, 70.6, 70.5, 34.9, 34.1, 31.6, 27.2) ppm.

Compound 23: HPLC: RT: 1.86 min, purity: 97.6%. LC-MS: RT=0.789 min,m/z=292.4 (M+H)⁺. ¹H NMR: (400 MHz, MeOD) δ 7.17-7.27 (m, 5H), 3.66-3.70(m, 4H), 3.49-3.64 (m, 2H), 2.84 (t, J=6.8, 2H), 2.37 (t, J=6.0, 2H),1.82-1.94 (m, 4H), 1.17-1.34 (m, 4H). ¹³C NMR: (400 MHz, MeOD) δ (173,140, 130, 129, 127, 73.0, 70.5, 68.1, 37.9, 37.2, 34.9, 31.5) ppm.

Compound 24: HPLC: RT: 2.24 min, purity: 98.3%. LC-MS: RT=0.825 min,m/z=292.1 (M+H)⁺. ¹H NMR: (400 MHz, MeOD) δ 7.24-7.28 (m, 2H), 7.15-7.20(m, 3H), 3.88 (s, 2H), 3.68-3.70 (m, 1H), 3.53-3.56 (m, 1H), 3.50 (t,J=6.4 Hz, 2H), 2.70 (t, J=7.6 Hz, 2H), 1.91-1.96 (m, 4H), 1.86-1.87 (m,2H), 1.33-1.38 (m, 4H). ¹³C NMR: (400 MHz, MeOD) δ (172, 143, 130, 129,127, 72.1, 71.1, 70.4, 34.9, 33.3, 32.3, 31.4) ppm.

Compound 25: HPLC: RT: 2.54 min, purity: 99.7%. LC-MS: RT=0.886 min,m/z=292.2 (M+H)⁺. ¹H NMR: (400 MHz, MeOD) δ 7.12-7.26 (m, 5H), 4.02 (t,J=6.0, 2H), 3.47-3.52 (m, 1H), 3.33-3.35 (m, 1H), 2.63 (t, J=7.2, 2H),1.88-1.94 (m, 4H), 1.64-1.66 (m, 4H), 1.1.23-1.31 (m, 4H). ¹³C NMR: (400MHz, MeOD) δ (158, 143, 130, 129, 126, 70.5, 65.6, 50.7, 36.5, 34.9,31.9, 29.9, 29.1) ppm.

Example 4B: The Cyclic Cyclopropane Backbone Analogue ofTrans-Stereoisomer MiM 111 is Also Active

Compound 26 was synthesized similarly to Compound 15B (Example 2),except incorporating 2-(3-phenylpropyl)cyclopropane-1-carboxylic acidfor 6-phenylhexanoic acid 1.2. This compound had an EC₅₀ of 5.1 nm, Hand M plasma protein binding of 94.4% and 95.5%, H and M liver microsomestability T^(1/2) values of >145 minutes and 114.1 minutes, and a PAMPAassay value of 58.451 nm/s.

Example 5: In Vivo Pharmacological Profiling Shows that Trans-MiM 111(Compound 15B) is a Clinical Candidate for CMT2A

Trans-MiM 111 exhibited high microsomal stability and passivepermeability (PAMPA Pe), with a low efflux ratio in NIH MDR1 cells thatdid not change in the presence of the P-gp inhibitor GF120918 (Table 3),indicating that it is not a P-gp substrate. In this context it wasanticipated that microsomal stability would correlate with in vivoplasma t^(1/2), and passive permeability with CNS levels.

TABLE 3 P-gp studies of trans-MiM 111 (Compound 15B) Mean P_(app) MeanCompound GF120918 (10⁻⁶ cm/s) Efflux Recovery % ID (+/−) A to B B to ARatio A to B B to A Note Nadolol − 0.26 ND ND 97.88 ND Low permeabilitymarker Metoprolol − 21.24 ND ND 105.27 ND High permeability markerDigoxin − 0.11 13.36 123.06 98.93 103.29 P-gp + 0.73 2.05 2.81 97.0499.34 substrate 15B − 20.36 35.45 1.74 84.58 100.21 — + 31.88 23.21 0.7384.76 100.61

This notion was examined by in vivo PK studies in mice, the only speciesin which pre-clinical models of human CMT2A have been published(Cartoni, R., et al.; Brain 133:1460-9, 2010; Bannerman, P., et al.;PLoS ONE 11:e0167573, 2016; Zhou, Y., et al.; J Clin Invest130:1756-1771, 2019). A target therapeutic brain level of 10 times thein vitro EC₅₀ for each compound, or 30 ng/g (˜100 nM), was established.

Comparative mouse plasma and brain levels were determined at increasingtimes after a single 10 mg/kg intravenous (IV) dose. The Vdss (volume ofdistribution at steady state) of trans-MiM 111 was 0.35 L/kg and peakbrain levels were 2,793 ng/g; therapeutic levels (of >30 ng/g) weremaintained for more than 2 hours after the single IV dose (FIG. 4A).

Because of its low Vdss, high Pe, and low efflux in NIH MDR1 cells itwas surmised that trans-MiM 111 might accumulate in brains over time. Ifthis were so, then brain levels after single dose administration couldbe misleading vis-à-vis therapeutic efficacy. To test this idea osmoticmini-pumps were used to deliver trans-MiM 111 subcutaneously (SQ) at adaily dose of 60 mg/kg/day for three days to achieve steady state, andits elimination kinetics were defined after mini-pump removal (FIG. 4B).Trans-MiM 111 plasma half-lives were similar after bolus IV and chronicSQ administration (1.1 hours and 1.33 hours, respectively), but itsbrain half-life was substantially longer after chronic infusion (3.37hours vs 1.06 hours after IV). Because it is 96.7% plasma protein boundin mice, the unbound fraction (fu) of trans-MiM 111 is 0.033. Using thisfactor to convert plasma [Cpd 15B]_(total) to plasma [Cpd15B]_(free/unbound), the calculated ratio of brain to free plasma levelis 10.8 after chronic infusion.

Example 6: In Vivo Trans-MiM 111 Engages its Mitochondrial Targets in aPre-Clinical Mouse Model of CMT2A

Taken together, the above results indicated that trans-MiM 111 hasproperties enabling it to activate mitofusins of neuronal mitochondriain CMT2A mice in vivo. In the only published study a prototype mitofusinactivator, Chimera B-A/I, increased mitochondrial motility whentopically applied to sciatic nerve neurons ex vivo (Rocha, A. G., etal.; Science 360:336-41, 2018). The in vivo effects of mitofusinactivators on CMT2A neuronal mitochondria were never assessed becausethis chemical class of mitofusin agonists has a very short in vivoplasma half-life of ˜0.2 hour. Because trans-MiM 111 is >75% orallybioavailable (FIG. 5A) its ability to engage peripheral nervemitochondrial targets in vivo was assessed after a single oral dose.Transgenic mice expressing the human MFN2 T105M mutation in motorneurons (Rocha A. G., et al.; Science 360:336-41, 2018) received 50mg/kg of trans-MiM 111 and mitochondrial motility in sciatic nerveneurons was assessed by a blinded investigator 6 hours thereafter.trans-MiM 111 markedly increased both the number and velocity of motilemitochondria in CMT2A mouse neurons (FIG. 5B).

Example 7: Off-Target, Specificity, and Safety Studies Show thatTrans-MiM 111 has Properties of an Advanced Clinical Lead for CMT2A

Off-target, specificity, and safety studies performed to determine thesuitability of trans-MiM 111 for possible clinical translation (Table 4)demonstrated that it is a potent and selective mitofusin activator witha favorable drug profile. It exhibited sub-10 nM potency for both MFN1and MFN2, with very low inhibitory activity for cytochrome P450 enzymes,indicating a small likelihood for drug-drug interactions. Activityscreening against hERG, hNAV1.5, hKCNQ, and a panel of 42receptors/kinases revealed only mild inhibition of dopamine aminotransferase (DAT) and monoamine oxidase (MAO-A; ˜30% inhibition at 10μM), indicating a safety window of 1,000-fold compared to on-targetefficacy, and a correspondingly limited potential for off-targetside-effects.

TABLE 4 Summary of key properties of trans-MiM 111 (Compound 158)On-target potency EC₅₀ Mfn1 KO cells (nm) 7.7 EC₅₀ Mfn2 KO cells (nm)9.5 Physical properties MW/cLogP/TPSA 289/3.22/49.33 Å kineticsolubility (mM) 175 fsp3 (%) 61 Selectivity and safety profiles hNav1.5(% inhib @ 10 mM) 8.3 hKCNQ (% inhib @ 10 mM) 0 hERG patch clamp IC₅₀(mM) >30 44 receptor/kinase panel (10 mM) DAT and MAO-A > 30% inhibCYP1A2/209/2019/2D6/3A4-M >50/>50/>50/>50/>50 IC₅₀ (mM) AMES testnegative

Materials and Methods Cell Lines

Wild-type MEFs were prepared from E10.5 c57/bl6 mouse embryos. SV-40 Tantigen-immortalized MFN1 null (CRL-2992), MFN2 null (CRL-2993) andMFN1/MFN2 double null MEFs (CRL-2994) were purchased from ATCC. MEFswere subcultured in DMEM (4.5 g/L glucose) plus 10% fetal bovine serum,1× nonessential amino acids, 2 mM L-glutamine, 100 units/mL penicillinand 100 μg/mL streptomycin.

Confocal Live Cell Studies of Mitochondria

Live cell imaging was performed on an Olympus Diaphot 200 fluorescencemicroscope equipped with a 60× water immersion objective. All live cellswere grown on coated glass-bottom 12-well plates and studied in modifiedKrebs-Henseleit buffer (138 mM NaCl, 3.7 mM KCl, 1.2 mM KH₂PO₄, 15 mM,20 mM HEPES and 1 mM CaCl₂) at room temperature.

Cells were excited with 408 nm (Hoechst), 561 nm (MitoTracker Green andCalcein AM, GFP), or 637 nm (TMRE, MitoTracker Orange, Ethidiumhomodimer-1, and AF594-Dextran) laser diodes. For mitochondrialelongation studies, mitochondrial aspect ratio (long axis/short axis)was calculated using automated edge detection and Image J software.Mitochondrial depolarization was calculated as percent of greenmitochondria visualized on MitoTracker Green and TMRE merged images,expressed as green/(green+yellow mitochondria)×100.

Preparative HPLC

Purification was performed using HPLC (H₂O-MeOH; Agilent 1260 Infinitysystems equipped with DAD and mass-detectors. Waters SunFire C18 OBDPrep Column, 100 Å, 5 μm, 19 mm×100 mm with SunFire C18 Prep GuardCartridge, 100 Å, 10 μm, 19 mm×10 mm) was used for separation. Thematerial was dissolved in 0.7 mL DMSO. Flow rate: 30 mL/minute. Purityof the obtained fractions was checked via analytical LCMS. Spectra wererecorded for each fraction as it was obtained straight afterchromatography in the solution form. The solvent was evaporated in theflow of N₂ at 80° C. On the basis of post-chromatography LCMS analysis,fractions were combined united. Solid fractions were dissolved in 0.5 mLMeOH and transferred into pre-weighted marked vials.

Obtained solutions were again evaporated in the flow of N₂ at 80° C.After drying, products were characterized by LCMS, ¹H NMR, and ¹³C NMR.

HPLC/HRMS (ESI)

LC/MS analysis was carried out using Agilent 1100 Series LC/MSD systemwith DAD\ELSD and Agilent LC\MSD VL (G1956 Å), SL (G1956B)mass-spectrometer or Agilent 1200 Series LC/MSD system with DAD\ELSD andAgilent LC\MSD SL (G6130 Å), SL (G6140 Å) mass-spectrometer. All theLC/MS data were obtained using positive/negative mode switching. Thecompounds were separated using a Zorbax SB-C18 1.8 μm 4.6×15 mm RapidResolution cartridge (PN 821975-932) under a mobile phase (A—ACN, 0.1%formic acid; B—water (0.1% formic acid)). Flow rate: 3 mL/minute;Gradient 0 minutes—100% B; 0.01 minute—100% B; 1.5 minutes—0% B; 1.8minutes—0% B; 1.81 minutes—100% B; Injection volume 1 μL; Ionizationmode atmospheric pressure chemical ionization (APCI); Scan range m/z80-1000.

Statistical Methods

Time-course and dose-response data are calculated for each study usingGraphPad Prism. All data are reported as mean±SEM. Statisticalcomparisons (two-sided) used one-way ANOVA and Tukey's tests formultiple groups or Student's t-test for paired comparisons. p<0.05 wasconsidered significant. In vitro pharmacokinetic analyses of mitofusinactivators was performed at WuXi Apptec Co. Ltd.

Binding to human and CD-1 mouse plasma proteins was measured usingequilibrium dialysis. Pooled individual frozen EDTA anticoagulatedplasma mouse and human samples were used as test matrix. Warfarin wasused as a positive control. The test compounds were spiked into blankmatrix at the final concentration of 2 μM. A 150-μL aliquot of matrixsample was added to one side of the chamber in a 96-well equilibriumdialyzer plate (HTD dialysis) and an equal volume of dialysis buffer wasadded to the other side of the chamber. An aliquot of matrix sample washarvested before the incubation and used as T₀ samples for recoverycalculation. The incubations were performed in triplicate. The dialyzerplate was placed in a humidified incubator and rotated slowly for fourhours at 37° C. After incubation, the samples were taken from the matrixside as well as the buffer side. The plasma sample was matched withequal volume of blank buffer; and buffer samples were matched with equalvolume of blank plasma. The matrix-matched samples were quenched withstop solution containing internal standard. All samples were analyzed byLC-MS/MS. All test compound concentrations in matrix and buffer samplesare expressed as peak area ratios (PAR) of analyte/internal standard.

In vitro stability was measured in human and mouse liver microsomes. Anintermediate solution (100 μM of small molecule) was initially preparedin methanol and subsequently used to prepare the working solution. Thiswas achieved by a 10-fold dilution step of the intermediate solution in100 mM potassium phosphate buffer. Ten microliters of a compound workingsolution or control working solution was added to all wells of a 96-wellplate for the time points (minutes): T₀, T₅, T₁₀, T₂₀, T₃₀, T₆₀, NCF60,except the matrix blank. The microsome solution (680 μL/well) (#452117,Corning; Woburn, Mass., USA; #R1000, Xenotech; Kansas City, Kans., USAand #M1000, Xenotech; Kansas City, Kans., USA) was dispersed to 96-wellplate as reservoir according to the plate map. Then, 80 μL/well wasadded to every plate by ADDA (Apricot Design Dual Arm, Apricot Designs,Inc., Covina, Calif., USA), and the mixture of microsome solution andcompound were allowed to incubate at 37° C. for about 10 minutes. Next,10 μL of 100 mM potassium phosphate buffer/well was added to NCF60 andincubated at 37° C. (timer 1H was started). After pre-warming, 90μL/well of NADPH (#00616, Sigma, Aldrich, St. Louis, Mo., USA)regenerating system was dispensed to 96-well plate as reservoiraccording to the plate map. Then 10 μL/well was added to every plate byADDA to start reaction. To terminate the reaction, 300 μL/well of stopsolution (cold in 4° C., including 100 ng/mL tolbutamide and 100 ng/mLlabetalol as internal standards) was used, and sampling plates wereagitated for approximately 10 minutes. The samples were next centrifugedat 4000 rpm for 20 minutes at 4° C. Supernatants were analyzed byLC-MS/MS.

Parallel Artificial Membrane Permeability Assay (PAMPA)

A 10 μM solution of a small molecule in 5% DMSO (150 μL) was added toeach well of the donor plate, whose PVDF membrane was pre-coated with 5μL of 1% brain polar lipid extract (porcine)/dodecane mixture. Then, 300μL of PBS was added to each well of the PTFE acceptor plate. The donorplate and acceptor plate were combined together and incubated for 4hours at room temperature with shaking at 300 rpm. To prepare the T₀sample, 20 μL of a donor solution was transferred to new well, followedby the addition of 250 μL PBS (DF:13.5) and 130 μL of ACN (containinginternal standard) as the T₀ sample. To prepare the acceptor sample, theplate was removed from incubator and 270 μL of the solution wastransferred from each acceptor well and mixed with 130 μL ACN(containing internal standard) as an acceptor sample. To prepare thedonor sample, 20 μL of the solution was transferred from each donor welland mixed with 250 μL PBS (DF: 13.5), 130 μL ACN (containing internalstandard) as a donor sample. The acceptor samples and donor samples wereanalyzed by LC-MS/MS.

The present invention is also directed to the following clauses.

Clause 1: A method of treating a peripheral nervous system (PNS) orcentral nervous system (CNS) genetic disorder, physical damage, and/orchemical injury, comprising: administering to a subject atherapeutically effective amount of a composition comprising one or moreof a trans-stereoisomer 6-phenylhexanamide derivative mitofusinactivator or a pharmaceutically acceptable salt thereof, wherein thetrans-stereoisomer 6-phenylhexanamide derivative mitofusin activatorstimulates mitochondrial fusion, increases mitochondrial fitness, andenhances mitochondrial subcellular transport.

Clause 2. The method of clause 1, wherein the composition comprises oneor more mitofusin activators, wherein the mitofusin activator comprisesa structure of formula:

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof,

wherein R¹ is a non-, mono-, or poly-substituted C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ aryl, or C₃₋₈ heterocyclyl.

Clause 3: The method of any of clauses 1 to 2, wherein the mitofusinactivator comprises a structure of formula:

and wherein R¹ is

Clause 4. The method of any of clauses 1 to 3, wherein R¹ isindependently and optionally substituted by one or more of acetamide,C₁₋₈ alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano,C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/or thiophene;wherein R¹ is optionally further substituted with one or more acetamide,alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfone, sulfur dioxide, and/orthiophene; and wherein one or more of the alkyl, cycloalkyl, heteroaryl,heterocyclyl, indole, or phenyl substituent is optionally furthersubstituted with one or more of the following substituents: acetamide,alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and thiophene.

Clause 5. The method of any of clauses 1 to 4, wherein the mitofusinactivator is:

Clause 6. A compound of Formula II

or a pharmaceutically acceptable salt thereof, wherein

Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R² and R³ are independently selected from H, F, alkyl, and C₃₋₇cycloalkyl, or R² and R³ are taken together to form a C₃₋₇ cycloalkyl orheterocycloalkyl;

R⁴ and R⁵ are independently selected from H, F, alkyl, and C₃₋₇cycloalkyl or R⁴ and R⁵ are taken together to form a C₃₋₇ cycloalkyl orheterocycloalkyl;

Y is O, CR⁶R⁷, CR⁸═CR⁹, a triple bond, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, NR⁸, S, SO₂, SONR⁹, —NR⁹SO₂—, —NR⁸CO—, —CONR⁸—, or—NR⁸CONR⁹—;

R⁶ is H, F, alkyl, or cycloalkyl; and R⁷ is H, F, alkyl, or cycloalkyl;or R⁶ and R⁷ are taken together to form C₃₋₇ cycloalkyl orheterocycloalkyl;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl;

R⁹ is H, alkyl, or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3, 4 or 5;

p is 0 or 1; and

q is 0, 1, 2, 3, 4 or 5, wherein when o is equal to or greater than 1,then Y=NR⁸, S, SO₂, SONR⁹, —NR⁹SO₂—, —NR⁸CO—, —CONR⁸—, —NR⁸CONR⁹—, andthe sum of o+p+q is not less than 3 or greater than 7.

Clause 7. The compound of clause 6, or a pharmaceutically acceptablesalt thereof, wherein

Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

Y is O, CR⁶R⁷, cycloalkyl, or aryl;

R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from H andalkyl;

o is 0, 1, 2, 3, 4 or 5;

p is 0 or 1; and

q is 0, 1, 2, 3, 4 or 5;

wherein when o is equal to or greater than 1, then X is S or SO₂; and

wherein the sum of o+p+q is not less than 3 or greater than 7.

Clause 8. The compound of any of clauses 6 to 7, or a pharmaceuticallyacceptable salt thereof, wherein

Z is aryl or heteroaryl;

Y is O, CH₂, or cycloalkyl;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl;

R⁹ is or H, alkyl, and C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3, 4 or 5;

p is 0 or 1; and

q is 0, 1, 2, 3, 4 or 5;

wherein when o is equal to or greater than 1, then X is S or SO₂; and

wherein the sum of o+p+q is not less than 3 or greater than 5.

Clause 9. The compound of any of clauses 6 to 8, or a pharmaceuticallyacceptable salt thereof, wherein

Z is aryl or heteroaryl;

Y is cyclopropyl or cyclobutyl;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl;

R⁹ is H, alkyl, COR⁷, or C₃₋₇ cycloalkyl;

or R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

o is 0, 1, 2, or 3;

p is 1; and

q is 0, 1, 2, or 3;

wherein the sum of o+p+q is not less than 3 or greater than 5.

Clause 10. The compound of any of clauses 6 to 9, or a pharmaceuticallyacceptable salt thereof, wherein

Z is aryl or heteroaryl;

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl; R⁹ is H, alkyl, COR⁷ or C₃₋₇cycloalkyl; or

R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 11. The compound of any of clauses 6 to 10, or a pharmaceuticallyacceptable salt thereof, wherein

Z is aryl or heteroaryl;

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 12. The compound of any of clauses 6 to 11, or a pharmaceuticallyacceptable salt thereof, wherein

Z is phenyl or heteroaryl; wherein the heterocyclic moiety contains 1 to4 atoms independently selected from nitrogen, oxygen and sulfur, andwherein the phenyl or heterocyclic moiety has 0 to 4 substituentsindependently selected from R⁸, OR⁸, Cl, F, —CN, CF₃, —NR⁸R⁹, —SO₂NR⁸R⁹,—NR⁸SO₂R⁹, —SO₂R⁹, —CONR⁸R¹⁰, —NR⁸COR¹⁰, C₃₋₇ cycloalkyl, andheterocycloalkyl;

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl; R⁹ is H, alkyl, COR⁷ or C₃₋₇cycloalkyl; or R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

R¹⁰ is independently selected from alkyl or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 13. The compound of any of clauses 6 to 12, or a pharmaceuticallyacceptable salt thereof, wherein

Z is phenyl or heteroaryl; wherein the heterocyclic moiety contains 1 to3 atoms independently selected from nitrogen, oxygen and sulfur, andwherein the phenyl or heterocyclic moiety has 0 to 3 substituentsindependently selected from R⁸, OR⁸, Cl, F, —CN, CF₃, —NR⁸R⁹, —SO₂R⁹,—CONR⁸R⁹, —NR⁷COR¹⁰, C₃₋₇ cycloalkyl, and heterocycloalkyl;

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl; R⁹ is H, alkyl, COR⁷ or C₃₋₇cycloalkyl; or R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

R¹⁰ is alkyl or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 14. The compound of any of clauses 6 to 13, or a pharmaceuticallyacceptable salt thereof, wherein

Z is phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 6-pyrimidinyl,5-pyrimidinyl, 4-pyrimidinyl or 2-pyrimidinyl, wherein the phenyl,pyridinyl, and pyrimidinyl moiety has 0 to 2 substituents independentlyselected from R⁸, OR⁸, Cl, F, —CN, CF₃, —NR⁸R⁹, —SO₂R¹⁰, —CONR⁸R⁹, and—NR⁸COR¹⁰

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl;

R⁹ is H, alkyl, COR⁷ or C₃₋₇ cycloalkyl; or

R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

R¹⁰ is alkyl or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 15. The compound of any of clauses 6 to 14, or a pharmaceuticallyacceptable salt thereof, wherein

Z is phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 6-pyrimidinyl,5-pyrimidinyl, 4-pyrimidinyl or 2-pyrimidinyl, wherein the phenyl,pyridinyl, and pyrimidinyl moiety has 0 to 2 substituents independentlyselected from R⁸, OR⁸, Cl, F, —CN, CF₃, —NR⁸R⁹, —SO₂R¹⁰, —CONR⁸R⁹, and—NR⁸COR¹⁰

Y is O or CH₂;

R², R³, R⁴, and R⁵ are CH₂;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl; R⁹ is H, alkyl, COR⁷ or C₃₋₇cycloalkyl; or

R⁸ and R⁹ are taken together to form C₃₋₇ cycloalkyl;

R¹⁰ is alkyl or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3 or 4;

p is 1; and

q is 0, 1, 2, 3 or 4;

wherein the sum of o+p+q is 5.

Clause 16. The compound of any of clauses 6 to 15, or pharmaceuticallyacceptable salt thereof, wherein the compound is

Clause 17. The compound of any of clauses 6 to 15, or pharmaceuticallyacceptable salt thereof, wherein the compound is:

Clause 18. A method of treating a disease for which a mitofusinactivator is indicated, the method comprising administering to a mammalin need thereof a therapeutically effective amount of a compound ofFormula II

or a pharmaceutically acceptable salt thereof, wherein

Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R² and R³ are independently selected from H, F, alkyl, and C₃₋₇cycloalkyl, or R² and R³ are taken together to form a C₃₋₇ cycloalkyl orheterocycloalkyl;

R⁴ and R⁵ are independently selected from H, F, alkyl, and C₃₋₇cycloalkyl or R⁴ and R⁵ are taken together to form a C₃₋₇ cycloalkyl orheterocycloalkyl;

Y is O, CR⁶R⁷, CR⁸═CR⁹, a triple bond, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, NR⁸, S, SO₂, SONR⁹, —NR⁹SO₂—, —NR⁸CO—, —CONR⁸—, or—NR⁸CONR⁹—;

R⁶ is H, F, alkyl, or cycloalkyl; and R⁷ is H, F, alkyl, or cycloalkyl;or R⁶ and R⁷ are taken together to form C₃₋₇ cycloalkyl orheterocycloalkyl;

R⁸ is H, alkyl, or C₃₋₇ cycloalkyl;

R⁹ is H, alkyl, or C₃₋₇ cycloalkyl;

o is 0, 1, 2, 3, 4 or 5;

p is 0 or 1; and

q is 0, 1, 2, 3, 4 or 5, wherein when o is equal to or greater than 1,then Y=NR⁸, S, SO₂, SONR⁹, —NR⁹SO₂—, —NR⁸CO—, —CONR^(B)—, —NR⁸CONR⁹—,and the sum of o+p+q is not less than 3 or greater than 7.

Clause 19. The method of any of clauses 1-5 or 18, wherein the PNS orCNS disorder is selected from any one or a combination of:

a chronic neurodegenerative condition wherein mitochondrial fusion,fitness, or trafficking are impaired;

a disease or disorder associated with mitofusin 1 (MFN1) or mitofusin 2(MFN2) dysfunction;

a disease associated with mitochondrial fragmentation, dysfunction, ordysmotility;

a degenerative neuromuscular condition such as Charcot-Marie-Toothdisease, Amyotrophic Lateral Sclerosis, Huntington's disease,Alzheimer's disease, Parkinson's disease;

hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);

diabetic neuropathy;

chemotherapy-induced peripheral neuropathy; and/or

crush injury, spinal cord injury (SCI), traumatic brain injury, stroke,optic nerve injury, and related conditions that involve axonaldisconnection.

Clause 20. The method of any of clauses 1-5, 18 or 19, with the provisothat the mitofusin activator is not selected from the followingcompounds:

Clause 21. The method of any of clauses 1-5 or 18-20, wherein thecomposition further comprises a pharmaceutically acceptable excipient.

Clause 22. A method of treating a CNS or PNS genetic or non-geneticneurodegenerative condition, injury, damage, or trauma comprisingadministering to the subject a therapeutically effective amount of thecompound of any one of clauses 6 to 17.

Clause 23. The method of clause 22, wherein the subject is diagnosedwith or is suspected of having:

a chronic neurodegenerative condition wherein mitochondrial fusion,fitness, or trafficking are impaired;

a disease or disorder associated with mitofusin 1 (MFN1) or mitofusin 2(MFN2) dysfunction;

a disease associated with mitochondrial fragmentation, dysfunction, ordysmotility;

a degenerative neuromuscular condition such as Charcot-Marie-Toothdisease, Amyotrophic Lateral Sclerosis, Huntington's disease,Alzheimer's disease, Parkinson's disease;

hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), Mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);

diabetic neuropathy;

chemotherapy-induced peripheral neuropathy; and/or

crush injury, spinal cord injury, traumatic brain injury, stroke, opticnerve injury, and related conditions that involve axonal disconnection.

Clause 24. A composition comprising the compound of any one of clauses 6to 17 and a pharmaceutically acceptable excipient.

The present disclosure is also directed to the following embodiments:

A. Methods for treating a mitochondria-associated disease, disorder, orcondition. The methods comprise: administering a therapeuticallyeffective amount of a composition comprising one or more of mitofusinactivator or a pharmaceutically acceptable salt thereof to a subjecthaving or suspected of having a mitochondria-associated disease,disorder or condition, the mitofusin activator having a structurerepresented by the following formula:

wherein:

-   -   Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;    -   R² and R³ are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl, or R² and R³ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   R⁴ and R⁵ are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl, or R⁴ and R⁵ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   Y is O, CR⁶R⁷, CR⁸═CR⁹, C≡C, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, NR⁸, S, SO₂, SONR⁹, NR⁹SO₂, NR⁸CO, CONR⁸, or        NR⁸CONR⁹;    -   R⁶ is H, F, alkyl, or cycloalkyl, R⁷ is H, F, alkyl, or        cycloalkyl, or R⁶ and R⁷ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   R⁸ is H, alkyl, or C₃₋₇ cycloalkyl, R⁹ is H, alkyl, or C₃₋₇        cycloalkyl, or R⁸ and R⁹ taken together form a C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4 or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4 or 5, provided that if Y is cycloalkyl and p        is 1 the sum of o+p+q is not less than 3 or greater than 5, and        otherwise the sum of o+p+q is 5.

B. Compositions comprising a mitofusin activator or a pharmaceuticallyacceptable salt thereof. The compositions comprise: a mitofusinactivator or a pharmaceutically acceptable salt thereof, the mitofusinactivator having a structure represented by the following formula:

wherein:

-   -   Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;    -   R² and R³ are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl, or R² and R³ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   R⁴ and R⁵ are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl, or R⁴ and R⁵ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   Y is O, CR⁶R⁷, CR⁸═CR⁹, C≡C, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, NR⁸, S, SO₂, SONR⁹, NR⁹SO₂, NR⁸CO, CONR⁸, or        NR⁸CONR⁹;    -   R⁶ is H, F, alkyl, or cycloalkyl, R⁷ is H, F, alkyl, or        cycloalkyl, or R⁶ and R⁷ taken together form a C₃₋₇ cycloalkyl        or heterocycloalkyl;    -   R⁸ is H, alkyl, or C₃₋₇ cycloalkyl, R⁹ is H, alkyl, or C₃₋₇        cycloalkyl, or R⁸ and R⁹ taken together form a C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4 or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4 or 5, provided that if Y is cycloalkyl and p        is 1 the sum of o+p+q is not less than 3 or greater than 5, and        otherwise the sum of o+p+q is 5.

Embodiments A and B may include one or more of the following elements:

Element 1: wherein:

Z is aryl or heteroaryl;

Y is O, CR⁶R⁷, or cycloalkyl;

R², R³, R⁴, R⁵, R⁶, and R⁷ are independently selected from H and alkyl;and

p is 1.

Element 2: wherein:

Y is cyclopropyl or cyclobutyl;

R², R³, R⁴ and R⁵ are H;

o is 0, 1, 2, or 3;

p is 1; and

q is 0, 1, 2 or 3.

Element 3: wherein:

Y is O or CH₂;

R², R³, R⁴ and R⁵ are H; and

p is 1.

Element 4: wherein Z is phenyl or heteroaryl, the heteroaryl containing1 to 4 atoms independently selected from nitrogen, oxygen and sulfur,and the phenyl or the heteroaryl having 0 to 4 substituentsindependently selected from R⁸, OR⁸, Cl, F, CN, CF₃, NR⁸R⁹, SO₂NR⁸R⁹,NR⁸SO₂R⁹, SO₂R⁹, CONR⁸R¹⁰, NR⁸COR¹⁰, C₃₋₇ cycloalkyl, andheterocycloalkyl; wherein R¹⁰ is alkyl or C₃₋₇ cycloalkyl.

Element 5: Z is phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl,6-pyrimidinyl, 5-pyrimidinyl, 4-pyrimidinyl or 2-pyrimidinyl, Z having 0to 2 substituents independently selected from R⁸, OR⁸, Cl, F, CN, CF₃,NR⁸R⁹, SO₂R¹⁰, CONR⁸R⁹, and NR⁸COR¹⁰; wherein R¹⁰ is alkyl or C₃₋₇cycloalkyl.

Element 6: wherein the mitofusin activator comprises atrans-stereoisomer 6-phenylhexanamide derivative or a pharmaceuticallyacceptable salt thereof; wherein the mitofusin activator stimulatesmitochondrial fusion, increases mitochondrial fitness, and enhancesmitochondrial subcellular transport.

Element 7: wherein the mitofusin activator has a structure representedby the following formula:

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof; wherein R¹ is a non-, mono-, or poly-substituted C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ aryl, or C₃₋₈ heterocyclyl.

Element 8: wherein R¹ is

Element 9: wherein the mitofusin activator has a structure representedby one or more of the following formulas:

Element 10: wherein the mitochondria-associated disease, disorder orcondition is a peripheral nervous system (PNS) or central nervous system(CNS) genetic or non-genetic disorder, physical damage, and/or chemicalinjury.

Element 11: wherein the PNS or CNS disorder is selected from any one ora combination of a chronic neurodegenerative condition whereinmitochondrial fusion, fitness, or trafficking are impaired; a disease ordisorder associated with mitofusin 1 (MFN1) or mitofusin 2 (MFN2)dysfunction; a disease associated with mitochondrial fragmentation,dysfunction, or dysmotility; a degenerative neuromuscular condition suchas Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,Huntington's disease, Alzheimer's disease, Parkinson's disease;hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);diabetic neuropathy; chemotherapy-induced peripheral neuropathy; crushinjury, spinal cord injury (SCI), traumatic brain injury, stroke, opticnerve injury, and related conditions that involve axonal disconnection;and any combination thereof.

Element 12: wherein the composition further comprises a pharmaceuticallyacceptable excipient.

All documents described herein are incorporated by reference herein forpurposes of all jurisdictions where such practice is allowed, includingany priority documents and/or testing procedures to the extent they arenot inconsistent with this text. As is apparent from the foregoinggeneral description and the specific embodiments, while forms of thedisclosure have been illustrated and described, various modificationscan be made without departing from the spirit and scope of thedisclosure. Accordingly, it is not intended that the disclosure belimited thereby. For example, the compositions described herein may befree of any component, or composition not expressly recited or disclosedherein. Any method may lack any step not recited or disclosed herein.Likewise, the term “comprising” is considered synonymous with the term“including.” Whenever a method, composition, element or group ofelements is preceded with the transitional phrase “comprising,” it isunderstood that we also contemplate the same composition or group ofelements with transitional phrases “consisting essentially of,”“consisting of,” “selected from the group of consisting of,” or “is”preceding the recitation of the composition, element, or elements andvice versa. The term “and/or” as used in a phrase such as “A and/or B”herein is intended to include “A and B,” “A or B,” “A,” and “B.”Numerical ranges used herein include the numbers recited in the range.For example, the numerical range “from 1 wt % to 10 wt %” includes 1 wt% and 10 wt % within the recited range.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the present specification and associated claims areto be understood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the embodiments of the present disclosure. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claim, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Whenever a numerical range with a lower limit and an upper limit isdisclosed, any number and any included range falling within the range isspecifically disclosed. In particular, every range of values (of theform, “from about a to about b,” or, equivalently, “from approximately ato b,” or, equivalently, “from approximately a-b”) disclosed herein isto be understood to set forth every number and range encompassed withinthe broader range of values. Also, the terms in the claims have theirplain, ordinary meaning unless otherwise explicitly and clearly definedby the patentee. Moreover, the indefinite articles “a” or “an,” as usedin the claims, are defined herein to mean one or more than one of theelement that it introduces.

One or more illustrative embodiments are presented herein. Not allfeatures of a physical implementation are described or shown in thisapplication for the sake of clarity. It is understood that in thedevelopment of a physical embodiment of the present disclosure, numerousimplementation-specific decisions must be made to achieve thedeveloper's goals, such as compliance with system-related,business-related, government-related and other constraints, which varyby implementation and from time to time. While a developer's effortsmight be time-consuming, such efforts would be, nevertheless, a routineundertaking for one of ordinary skill in the art and having benefit ofthis disclosure.

Therefore, the present disclosure is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent disclosure may be modified and practiced in different butequivalent manners apparent to one having ordinary skill in the art andhaving the benefit of the teachings herein. Furthermore, no limitationsare intended to the details of construction or design herein shown,other than as described in the claims below. It is therefore evidentthat the particular illustrative embodiments disclosed above may bealtered, combined, or modified and all such variations are consideredwithin the scope and spirit of the present disclosure. The embodimentsillustratively disclosed herein suitably may be practiced in the absenceof any element that is not specifically disclosed herein and/or anyoptional element disclosed herein.

What is claimed is:
 1. A method comprising: administering atherapeutically effective amount of a composition comprising one or moremetabolites of trans-(4-hydroxycyclohexyl)-6-phenylhexanamide or apharmaceutically acceptable salt thereof to a subject having orsuspected of having a mitochondria-associated disease, disorder orcondition, the one or more metabolites being active to promote mitofusinactivation.
 2. The method of claim 1, wherein the one or moremetabolites are selected from the group consisting of

and any combination thereof.
 3. The method of claim 1, wherein themitochondria-associated disease, disorder or condition is a peripheralnervous system (PNS) or central nervous system (CNS) genetic ornon-genetic disorder, physical damage, and/or chemical injury.
 4. Themethod of claim 3, wherein the PNS or CNS disorder is selected from anyone or a combination of a chronic neurodegenerative condition whereinmitochondrial fusion, fitness, or trafficking are impaired; a disease ordisorder associated with mitofusin 1 (MFN1) or mitofusin 2 (MFN2)dysfunction; a disease associated with mitochondrial fragmentation,dysfunction, or dysmotility; a degenerative neuromuscular condition suchas Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,Huntington's disease, Alzheimer's disease, Parkinson's disease;hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);diabetic neuropathy; chemotherapy-induced peripheral neuropathy; crushinjury, spinal cord injury (SCI), traumatic brain injury, stroke, opticnerve injury, and related conditions that involve axonal disconnection;and any combination thereof.
 5. A method comprising: administering atherapeutically effective amount of a composition comprisingtrans-(4-hydroxycyclohexyl)-6-phenylhexanamide or a pharmaceuticallyacceptable salt thereof to a subject having or suspected of having amitochondria-associated disease, disorder or condition; and forming oneor more metabolites of thetrans-(4-hydroxycyclohexyl)-6-phenylhexanamide in vivo within thesubject, the one or more metabolites being active to promote mitofusinactivation and being selected from the group consisting of

and any combination thereof.
 6. The method of claim 5, wherein themitochondria-associated disease, disorder or condition is a peripheralnervous system (PNS) or central nervous system (CNS) genetic ornon-genetic disorder, physical damage, and/or chemical injury.
 7. Themethod of claim 6, wherein the PNS or CNS disorder is selected from anyone or a combination of a chronic neurodegenerative condition whereinmitochondrial fusion, fitness, or trafficking are impaired; a disease ordisorder associated with mitofusin 1 (MFN1) or mitofusin 2 (MFN2)dysfunction; a disease associated with mitochondrial fragmentation,dysfunction, or dysmotility; a degenerative neuromuscular condition suchas Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,Huntington's disease, Alzheimer's disease, Parkinson's disease;hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);diabetic neuropathy; chemotherapy-induced peripheral neuropathy; crushinjury, spinal cord injury (SCI), traumatic brain injury, stroke, opticnerve injury, and related conditions that involve axonal disconnection;and any combination thereof.
 8. A composition comprising: one or moremetabolites of trans-(4-hydroxycyclohexyl)-6-phenylhexanamide or apharmaceutically acceptable salt thereof, the one or more metabolitesbeing active to promote mitofusin activation.
 9. The composition ofclaim 8, wherein the one or more metabolites are selected from the groupconsisting of

and any combination thereof.
 10. The composition of claim 8, furthercomprising: a pharmaceutically acceptable excipient.